Polynucleotide compositions encoding broad spectrum delta-endotoxins

ABSTRACT

Disclosed are novel synthetically-modified  B. thuringiensis  chimeric crystal proteins having improved insecticidal activity against coleopteran, dipteran and lepidopteran insects. Also disclosed are the nucleic acid segments encoding these novel peptides. Methods of making and using these genes and proteins are disclosed as well as methods for the recombinant expression, and transformation of suitable host cells. Transformed host cells and transgenic plants expressing the modified endotoxin are also aspects of the invention.

This application is a division of application Ser. No. 09/253,341 filedFeb. 19, 1999, now U.S. Pat. No. 6,242,241, which is a division ofapplication Ser. No. 08/922,505 filed Sep. 3, 1997, now U.S. Pat. No.6,110,464, which is a continuation-in-part of application Ser. No.08/754,490 filed Nov. 20, 1996, now U.S. Pat. No. 6,017,534, the entirecontents of which is incorporated by reference herein.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention provides new proteins for combatting insects, andparticularly, coleopteran, dipteran, and lepidopteran insects sensitiveto the disclosed δ-endotoxins derived from Bacillus thuringiensis. Theinvention provides novel chimeric crystal proteins and the chimeric crygene segments which encode them, as well as methods for making and usingthese DNA segments, methods of producing the encoded proteins, methodsfor making synthetically-modified chimeric crystal proteins, and methodsof making and using the synthetic crystal proteins.

1.2 Description of Related Art

1.2.1 B. Thuringiensis Crystal Proteins

The Gram-positive soil bacterium B. thuringiensis is well known for itsproduction of proteinaceous parasporal crystals, or δ-endotoxins, thatare toxic to a variety of lepidopteran, coleopteran, and dipteranlarvae. B. thuringiensis produces crystal proteins during sporulationwhich are specifically toxic to certain species of insects. Manydifferent strains of B. thuringiensis have been shown to produceinsecticidal crystal proteins, and compositions comprising B.thuringiensis strains which produce proteins having insecticidalactivity have been used commercially as environmentally-acceptableinsecticides because of their toxicity to the specific target insect,and non-toxicity to plants and other non-targeted organisms.

Commercial formulations of naturally occurring B. thuringiensis isolateshave long been used for the biological control of agricultural insectpests. In commercial production, the spores and crystals obtained fromthe fermentation process are concentrated and formulated for foliarapplication according to conventional agricultural practices.

1.2.2. Nomenclature of Crystal Proteins

A review by Höfte et al., (1989) describes the general state of the artwith respect to the majority of insecticidal B. thuringiensis strainsthat have been identified which are active against insects of the OrderLepidoptera, i.e., caterpillar insects. This treatise also describes B.thuringiensis strains having insecticidal activity against insects ofthe Orders Diptera (ie. flies and mosquitoes) and Coleoptera (i.e.beetles). A number of genes encoding crystal proteins have been clonedfrom several strains of B. thuringiensis. Höfte et al. (1989) discussesthe genes and proteins that were identified in B. thuringiensis prior to1990, and sets forth the nomenclature and classification scheme whichhas traditionally been applied to B. thuringiensis genes and proteins.cry1 genes encode lepidopteran-toxic Cry1 proteins. cry2 genes encodeCry2 proteins that are toxic to both lepidopterans and dipterans. cry3genes encode coleopteran-toxic Cry3 proteins, while cry4 genes encodedipteran-toxic Cry4 proteins, etc.

Recently a new nomenclature has been proposed which systematicallyclassifies the Cry proteins based upon amino acid sequence homologyrather than upon insect target specificities. This classification schemeis summarized in Table 1.

TABLE 1 REVISED B. THURINGIENSIS δ-ENDOTOXIN NOMENCLATURE^(A) New OldGenBank Accession # Cry1Aa CryIA(a) M11250 Cry1Ab CryIA(b) M13898 Cry1AcCryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1Ae CryIA(e) M65252 Cry1BaCryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442 Cry1Bd CryE1 U70726Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160 Cry1DbPrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b) M73253 Cry1Fa CryIFM63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1Gb CryH2 U70725 Cry1HaPrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1Ib CryV U07642 Cry1JaET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2Aa CryIIA M31738 Cry2AbCryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIA M22472 Cry3Ba CryIIIBX17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797 Cry4A CryIVA Y00423Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry5BU19725 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIIC M64478Cry7Ab CryIIICb U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365 Cry8CCryIIIF U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527Cry10A CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902 Cry12ACryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34kDa M76442Cry16A cbm71 X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049 Cry19AJeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt1B U37196 Cyt2ACytB Z14147 Cyt2B CytB U52043 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

1.2.3 Mode of Crystal Protein Toxicity

All δ-endotoxin crystals are toxic to insect larvae by ingestion.Solubilization of the crystal in the midgut of the insect releases theprotoxin form of the δ-endotoxin which, in most instances, issubsequently processed to an active toxin by midgut protease. Theactivated toxins recognize and bind to the brush-border of the insectmidgut epithelium through receptor proteins. Several putative crystalprotein receptors have been isolated from certain insect larvae (Knightet al., 1995; Gill et al., 1995; Masson et al, 1995). The binding ofactive toxins is followed by intercalation and aggregation of toxinmolecules to form pores within the midgut epithelium. This process leadsto osmotic imbalance, swelling, lysis of the cells lining the midgutepithelium, and eventual larvae mortality.

1.2.4 Molecular Biology of δ-Endotoxins

With the advent of molecular genetic techniques, various δ-endotoxingenes have been isolated and their DNA sequences determined. These geneshave been used to construct certain genetically engineered B.thuringiensis products that have been approved for commercial use.Recent developments have seen new δ-endotoxin delivery systemsdeveloped, including plants that contain and express geneticallyengineered δ-endotoxin genes.

The cloning and sequencing of a number of δ-endotoxin genes from avariety of Bacillus thuringiensis strains have been described and aresummarized by Höfte and Whiteley, 1989. Plasmid shuttle vectors designedfor the cloning and expression of δ-endotoxin genes in E. coli or B.thuringiensis are described by Gawron-Burke and Baum (1991). U.S. Pat.No. 5,441,884 discloses a site-specific recombination system forconstructing recombinant B. thuringiensis strains containing δ-endotoxingenes that are free of DNA not native to B. thuringiensis.

The Cry1 family of crystal proteins, which are primarily active againstlepidopteran pests, are the best studied class of δ-endotoxins. Thepro-toxin form of Cry1 δ-endotoxins consist of two approximately equalsized segments. The carboxyl-half, or pro-toxin segment, is not toxicand is thought to be important for crystal formation (Arvidson et al.,1989). The amino-half of the protoxin comprises the active-toxin segmentof the Cry1 molecule and may be further divided into three structuraldomains as determined by the recently described crystallographicstructure for the active toxin segment of the Cry1Aa δ-endotoxin(Grochulski et al., 1995). Domain 1 occupies the first third of theactive toxin and is essential for channel formation (Thompson et al.,1995). Domain 2 and domain 3 occupy the middle and last third of theactive toxin, respectively. Both domains 2 and 3 have been implicated inreceptor binding and insect specificity, depending on the insect andδ-endotoxin being examined (Thompson et al., 1995).

1.2.5 Chimeric Crystal Proteins

In recent years, researchers have focused effort on the construction ofhybrid δ-endotoxins with the hope of producing proteins with enhancedactivity or improved properties. Advances in the art of moleculargenetics over the past decade have facilitated a logical and orderlyapproach to engineering proteins with improved properties. Site-specificand random mutagenesis methods, the advent of polymerase chain reaction(PCRT) methodologies, and the development of recombinant methods forgenerating gene fusions and constructing chimeric proteins havefacilitated an assortment of methods for changing amino acid sequencesof proteins, fusing portions of two or more proteins together in asingle recombinant protein, and altering genetic sequences that encodeproteins of commercial interest.

Unfortunately, for crystal proteins, these techniques have only beenexploited in limited fashion. The likelihood of arbitrarily creating achimeric protein with enhanced properties from portions of the numerousnative proteins which have been identified is remote given the complexnature of protein structure, folding, oligomerization, activation, andcorrect processing of the chimeric protoxin to an active moiety. Only bycareful selection of specific target regions within each protein, andsubsequent protein engineering can toxins be synthesized which haveimproved insecticidal activity.

Some success in the area, however, has been reported in the literature.For example, the construction of a few hybrid δ-endotoxins is reportedin the following related art: Intl. Pat. Appl. Publ. No. WO 95/30753discloses the construction of hybrid B. thuringiensis δ-endotoxins forproduction in Pseudomonas fluorescens in which the non-toxic protoxinfragment of Cry1F has been replaced by the non-toxic protoxin fragmentfrom the Cry1Ac/Cry1Ab that is disclosed in U.S. Pat. No. 5,128,130.

U.S. Pat. No. 5,128,130 discloses the construction of hybrid B.thuringiensis δ-endotoxins for production in P. fluorescens in which aportion of the non-toxic protoxin segment of Cry1Ac is replaced with thecorresponding non-toxic protoxin fragment of Cry1Ab. U.S. Pat. No.5,055,294 discloses the construction of a specific hybrid δ-endotoxinbetween Cry1Ac (amino acid residues 1466) and Cry1Ab (amino acidresidues 466-1155) for production in P. fluorescens. Although theaforementioned patent discloses the construction of a hybrid toxinwithin the active toxin segment, no specifics are presented in regard tothe hybrid toxin's insecticidal activity. Intl. Pat. Appl. Publ. No. WO95/30752 discloses the construction of hybrid B. thuringiensisδ-endotoxins for production in P. fluorescens in which the non-toxicprotoxin segment of Cry1C is replaced by the non-toxic protoxin segmentfrom Cry1Ab. The aforementioned application further discloses that theactivity against Spodoptera exigua for the hybrid δ-endotoxin isimproved over that of the parent active toxin, Cry1C.

Intl. Pat. Appl. Publ. No. WO 95/06730 discloses the construction of ahybrid B. thuringiensis δ-endotoxin consisting of domains 1 and 2 ofCry1E coupled to domain 3 and the non-toxic protoxin segment of Cry1C.Insect bioassays performed against Manduca sexta (sensitive to Cry1C andCry1E), Spodoptera exigua (sensitive to Cry1C), and Mamestra brassicae(sensitive to Cry1C) show that the hybrid Cry1E/Cry1C hybrid toxin isactive against M. sexta, S. exigua, and M. brassicae. The bioassayresults were expressed as EC₅₀ values (toxin concentration giving a 50%growth reduction) rather than LC₅₀ values (toxin concentration giving50% mortality). Although the δ-endotoxins used for bioassay wereproduced in B. thuringiensis, only artificially-generated activesegments of the δ-endotoxins were used, not the naturally-producedcrystals typically produced by B. thuringiensis that are present incommercial B. thuringiensis formulations. Bioassay results indicatedthat the LC₅₀ values for the hybrid Cry1E/Cry1C crystal against S.frugiperda were 1.5 to 1.7 Fold lower (more active) than for nativeCry1C. This art also discloses the construction of a hybrid B.thuringiensis δ-endotoxin between Cry1Ab (domains 1 and 2) and Cry1C(domain 3 and the non-toxic protoxin segment), although no data aregiven regarding the hybrid toxin's activity or usefulness.

Lee et al. (1995) report the construction of hybrid B. thuringiensisδ-endotoxins between Cry1Ac and Cry1Aa within the active toxin segment.Artificially generated active segments of the hybrid toxins were used toexamine protein interactions in susceptible insect brush bordermembranes vesicles (BBMV). The bioactivity of the hybrid toxins was notreported.

Honee et al. (1991) report the construction of hybrid δ-endotoxinsbetween Cry1C (domain 1) and Cry1Ab (domains 2 and 3) and the reciprocalhybrid between Cry1Ab (domain 1) and Cry1C (domains 2 and 3). Thesehybrids failed to show any significant increase in activity, againstsusceptible insects. Furthermore, the Cry1C (domain 1)/Cry1Ab (domains 2and 3) hybrid toxin was found to be hypersensitive to proteasedegradation. A report by Schnepf et al. (1990) discloses theconstruction of Cry1Ac hybrid toxin in which a small portion of domain 2was replaced by the corresponding region of Cry1Aa, although nosignificant increase in activity against susceptible insect larvae wasobserved.

1.3 Deficiencies in the Prior Art

The limited successes in producing chimeric crystal proteins which haveimproved activity have negatively impacted the field by thwartingefforts to produce recombinantly-engineered crystal protein forcommercial development, and to extend the toxic properties and hostspecificities of the known endotoxins. Therefore, what is lacking in theprior art are reliable methods and compositions comprisingrecombinantly-engineered crystal proteins which have improvedinsecticidal activity, broad-host-range specificities, and which aresuitable for commercial production in B. thuringiensis.

2. SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations in the priorart by providing novel chimeric δ-endotoxins which have improvedinsecticidal properties, and broad-range specificities.

Disclosed are methods for the construction of B. thuringiensis hybridδ-endotoxins comprising amino acid sequences from native Cry1Ac andCry1F crystal proteins. These hybrid proteins, in which all or a portionof Cry1Ac domain 2, all or a portion of Cry1Ac domain 3, and all or aportion of the Cry1Ac protoxin segment is replaced by the correspondingportions of Cry1F, possess not only the insecticidal characteristics ofthe parent δ-endotoxins, but also have the unexpected and remarkableproperties of enhanced broad-range specificity which is not proficientlydisplayed by either of the native δ-endotoxins from which the chimericproteins were engineered.

Specifically, the present invention discloses and claimsgenetically-engineered hybrid δ-endotoxins which comprise a portion of aCry1Ac crystal protein fused to a portion of a Cry1F crystal protein.These chimeric endotoxins have broad-range specificity for the insectpests described herein.

In a further embodiment, the present invention also discloses and claimsrecombinant B. thuringiensis hybrid δ-endotoxins which comprise aportion of Cry1Ab, Cry1F, and Cry1Ac in which all or a portion of Cry1Abdomain 2 or all or a portion of Cry1Ab domain 3 is replaced by thecorresponding portions of Cry1F and all or a portion of the Cry1Abprotoxin segment is replaced by the corresponding portions of Cry1Ac.Exemplary hybrid δ-endotoxins between Cry1Ab and Cry1F are identified inSEQ ID NO:13 and SEQ ID NO:14.

One aspect of the present invention demonstrates the unexpected resultthat certain hybrid δ-endotoxins derived from Cry1Ac and Cry1F proteinsexhibit not only the insecticidal characteristics of the parentδ-endotoxins, but also possess insecticidal activity which is notproficiently displayed by either of the parent δ-endotoxins.

Another aspect of the invention further demonstrates the unexpectedresult that certain chimeric Cry1Ab/Cry1F proteins maintain not only theinsecticidal characteristics of the parent δ-endotoxins, but alsoexhibit insecticidal activity which is not displayed by either thenative Cry1Ab or Cry1F endotoxins.

The present invention also encompasses Cry1Ac/Cry1F and Cry1Ab/Cry1Fhybrid δ-endotoxins that maintain the desirable characteristics neededfor commercial production in B. thuringiensis. Specifically, the hybridδ-endotoxins identified in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:34 can efficientlyform proteinaceous parasporal inclusions in B. thuringiensis and havethe favorable characteristics of solubility, protease susceptibility,and insecticidal activity of the parent δ-endotoxins.

In a further embodiment, the present invention also discloses and claimsrecombinant B. thuringiensis hybrid δ-endotoxins which comprise aportion of Cry1Ac and Cry1C in which all or a portion of Cry1Ac domain 3is replaced by the corresponding portions of Cry1C and all or a portionof the Cry1Ac protoxin segment is replaced by the corresponding portionof Cry1C. Exemplary hybrid δ-endotoxins between Cry1Ac and Cry1C areidentified in SEQ ID NO:29 and SEQ ID NO:30.

One aspect of the present invention demonstrates the unexpected resultthat, although neither Cry1Ac nor Cry1C possess S. frugiperda activity,the Cry1Ac/Cry1C hybrid δ-endotoxin identified by SEQ ID NO:29 and SEQID NO:30 has significant activity against S. frugiperda. Furthermore,the Cry1Ac/Cry1C hybrid δ-endotoxin identified by SEQ ID NO:29 and SEQID NO:30 has significantly better activity against S. exigua than theCry1C parental δ-endotoxin.

The present invention further pertains to the recombinant nucleic acidsequences which encode the novel crystal proteins disclosed herein.Specifically, the invention discloses and claims the nucleic acidsequences of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, and SEQ ID NO:33; nucleic acid sequences whichare complementary to the nucleic acid sequences of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29; and SEQID NO:33, and nucleic acid sequences which hybridize to the sequences ofSEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQID NO:29, and SEQ ID NO:33.

The novel hybrid δ-endotoxins disclosed herein are useful in the controlof a broad range of insect pests. These hybrid δ-endotoxins aredescribed in FIG. 1 and FIG. 4 and are disclosed in SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQID NO:34. The nucleic acid segments encoding these proteins aredisclosed in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, and SEQ ID NO:33. The insecticidal andbiochemical properties of the hybrid δ-endotoxins are described in FIG.2, FIG. 3, and Table 4, Table 5, Table 6, and Table 7. The broad hostrange of the improved δ-endotoxins specified in the present invention isuseful in circumventing dilution effects caused by expressing multipleδ-endotoxin genes within a single B. thuringiensis strain. Expression ofsuch a broad host range δ-endotoxin in plants is expected to impartprotection against a wider variety of insect pests.

The impetus for constructing these and other hybrid δ-endotoxins is tocreate novel toxins with improved insecticidal activity, increasedhost-range specificity, and improved production characteristics. The DNAsequences listed in Table 7 define the exchange points for the hybridδ-endotoxins pertinent to the present invention and as oligonucleotideprimers, may be used to identify like or similar hybrid δ-endotoxins bySouthern or colony hybridization under conditions of moderate to highstringency. Researchers skilled in the art will recognize the importanceof the exchange site chosen between two or more δ-endotoxins can beachieved using a number of in vivo or in vitro molecular genetictechniques. Small variations in the exchange region between two or moreδ-endotoxins may yield similar results or, as demonstrated for EG11062and EG11063, adversely affect desirable traits. Similarly, largevariations in the exchange region between two or more δ-endotoxins mayhave no effect on desired traits, as demonstrated by EG11063 andEG11074, or may adversely affect desirable traits, as demonstrated byEG11060 and EG11063.

Favorable traits with regard to improved insecticidal activity,increased host range, and improved production characteristics may beachieved by other such hybrid δ-endotoxins including, but not limitedto, the cry1, cry2, cry3, cry4, cry5; cry6, cry7, cry8, cry9, cry10,cry11, cry12, cry13, cry14, cry15 class of δ-endotoxin genes and the B.thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes ofB. thuringiensis insecticidal proteins include, but are not limited toCry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ba, Cry1Bb, Cry1Ca, Cry1Cb,Cry1Da, Cry1Db, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Ha, Cry2a,Cry2b, Cry1Ja, Cry1Ka, Cry11Aa, Cry11Ab, Cry12Aa, Cry3Ba, Cry3Bb, Cry3C,Cry4a, Cry4Ba, Cry5Sa, Cry5Ab, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry8Aa,Cry8Ba, Cry8Ca, Cry9Aa, Cry9Ba, Cry9Ca, Cry10Aa, Cry11Aa, Cry12Aa,Cry13Aa, Cry14Aa, Cry15Aa, Cyt1Aa, and Cyt2Aa. Related hybridδ-endotoxins would consist of the amino portion of one of theaforementioned δ-endotoxins, including all or part of domain 1 or domain2, Fused to all or part of domain 3 from another of the aforementionedδ-endotoxins. The non-active protoxin fragment of such hybridδ-endotoxins may consist of the protoxin fragment from any of theaforementioned δ-endotoxins which may act to stabilize the hybridδ-endotoxin as demonstrated by EG11087 and EG11091 (see e.g., Table 4).Hybrid δ-endotoxins possessing similar traits as those described in thepresent invention could be constructed by conservative, or “similar”replacements of amino acids within hybrid δ-endotoxins. Suchsubstitutions would mimic the biochemical and biophysical properties ofthe native amino acid at any position in the protein. Amino acidsconsidered similar include for example, but are not limited to:

Ala, Ser, and Thr;

Asp and Glu;

Asn and Gln;

Lys and Arg;

Ile, Leu, Met, and Val; and

Phe, Tyr, and Trp.

Researchers skilled in the art will recognize that improved insecticidalactivity, increased host range, and improved production characteristicsimparted upon hybrid δ-endotoxins may be further improved by alteringthe genetic code for one or more amino acid positions in the hybridδ-endotoxin such that the position, or positions, is replaced by anyother amino acid. This may be accomplished by targeting a region orregions of the protein for mutagenesis by any number of establishedmutagenic techniques, including those procedures relevant to the presentinvention. Such techniques include site-specific mutagenesis (Kunkle,1985; Kunkle et al., 1987), DNA shuffling (Stemmer, 1994), and PCR™overlap extension (Horton et al., 1989). Since amino acids situated ator near the surface of a protein are likely responsible for itsinteraction with other proteinaceous or non-proteinaceous moieties, theymay serve as “target” regions for mutagenesis. Such surface exposedregions may consist of, but not be limited to, surface exposed aminoacid residues within the active toxin fragment of the protein andinclude the inter-α-helical or inter-β-strand “loop”-regions ofδ-endotoxins that separate α-helices within domain 1 and β-strandswithin domain 2 and domain 3. Such procedures may favorably change theprotein's biochemical and biophysical characteristics or its mode ofaction as outlined in the Section 1. These include, but are not limitedto: 1) improved crystal formation, 2) improved protein stability orreduced protease degradation, 3) improved insect membrane receptorrecognition and binding, 4) improved oligomerization or channelformation in the insect midgut endothelium, and 5) improved insecticidalactivity or insecticidal specificity due to any or all of the reasonsstated above.

2.1 Crystal Protein Transgenes and Transgenic Plants

In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid segmentencoding the novel chimeric crystal proteins of the present invention.The process of producing transgenic plants is well-known in the art. Ingeneral, the method comprises transforming a suitable host cell with aDNA segment which contains a promoter operatively linked to a codingregion that encodes a B. thuringiensis Cry1Ac-1F or Cry1Ab-1F,Cry1Ac-1C, or a Cry1Ab-1Ac-1F chimeric crystal protein. Such a codingregion is generally operatively linked to a transcription-terminatingregion, whereby the promoter is capable of driving the transcription ofthe coding region in the cell, and hence providing the cell the abilityto produce the recombinant protein in vivo. Alternatively, in instanceswhere it is desirable to control, regulate, or decrease the amount of aparticular recombinant crystal protein expressed in a particulartransgenic cell, the invention also provides for the expression ofcrystal protein antisense mRNA. The use of antisense mRNA as a means ofcontrolling or decreasing the amount of a given protein of interest in acell is well-known in the art.

Another aspect of the invention comprises a transgenic plant whichexpress a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant” is intended to refer to a plant that has incorporatedDNA sequences, including but not limited to genes which are perhaps notnormally present, DNA sequences not normally transcribed into RNA ortranslated into a protein (“expressed”), or any other genes or DNAsequences which one desires to introduce into the non-transformed plant,such as genes which may normally be present in the non-transformed plantbut which one desires to either genetically engineer or to have alteredexpression. The construction and expression of synthetic B.thuringiensis genes in plants has been described in detail in U.S. Pat.Nos. 5,500,365 and 5,380,831 (each specifically incorporated herein byreference).

It is contemplated that in some instances the genome of a transgenicplant of the present invention will have been augmented through thestable introduction of one or more cry1Ac-1F, cry1Ab-1F, cry1Ac-1C, orcry1Ab-1Ac-1F transgenes, either native, synthetically-modified, orfurther mutated. In some instances, more than one transgene will beincorporated into the genome of the transformed host plant cell. Such isthe case when more than one crystal protein-encoding DNA segment isincorporated into the genome of such a plant. In certain situations, itmay be desirable to have one, two, three, four, or even more B.thuringiensis crystal proteins (either native orrecombinantly-engineered) incorporated and stably expressed in thetransformed transgenic plant.

A preferred gene, such as those disclosed in SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:33which may be introduced includes, for example, a crystalprotein-encoding a DNA sequence from bacterial origin, and particularlyone or more of those described herein which are obtained from Bacillusspp. Highly preferred nucleic acid sequences are those obtained from B.thuringiensis, or any of those sequences which have been geneticallyengineered to decrease or increase the insecticidal activity of thecrystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of a transgeniccell line are well-known in the art, and are discussed herein. Vectors,plasmids, cosmids, yeast artificial chromosomes (YACs) and nucleic acidsegments for use in transforming such cells will, of course, generallycomprise either the operons, genes, or gene-derived sequences of thepresent invention, either native, or synthetically-derived, andparticularly those encoding the disclosed crystal proteins. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even gene sequences which have positively- ornegatively-regulating activity upon the particular genes of interest asdesired. The DNA segment or gene may encode either a native or modifiedcrystal protein, which will be expressed in the resultant recombinantcells, and/or which will impart an improved phenotype to the regeneratedplant. Nucleic acid sequences optimized for expression in plants havebeen disclosed in Intl. Pat. Appl. Publ. No. WO 93/07278 (specificallyincorporated herein by reference).

Such transgenic plants may be desirable for increasing the insecticidalresistance of a monocotyledonous or dicotyledonous plant, byincorporating into such a plant, a transgenic DNA segment encodingCry1Ac-1F and/or Cry1Ac-1C, and/or Cry1Ab-1F and/or Cry1Ab-1Ac-1Fcrystal protein(s) which possess broad-insect specificity. Particularlypreferred plants such as grains, including but not limited to corn,wheat, oats, rice, maize, and barley; cotton; soybeans and otherlegumes; trees, including but not limited to ornamentals, shrubs,fruits, nuts; vegetables, turf and pasture grasses, berries, citrus, andother crops of commercial interest; such as garden crops and/orhouseplants, succulents, cacti, and flowering species.

In a related aspect, the present invention also encompasses a seedproduced by the transformed plant, a progeny from such seed, and a seedproduced by the progeny of the original transgenic plant, produced inaccordance with the above process. Such progeny and seeds will have astably crystal protein transgene stably incorporated into its genome,and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more chimeric crystal proteins or polypeptidesare aspects of this invention.

2.2 Crystal Protein Screening and Immunodetection Kits

The present invention contemplates methods and kits for screeningsamples suspected of containing crystal protein polypeptides or crystalprotein-related polypeptides, or cells producing such polypeptides.Exemplary proteins include those disclosed in SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQID NO:34. Said kit can contain a nucleic acid segment or an antibody ofthe present invention. The kit can contain reagents for detecting aninteraction between a sample and a nucleic acid or antibody of thepresent invention. The provided reagent can be radio-, fluorescently- orenzymatically-labeled. The kit can contain a known radiolabeled agentcapable of binding or interacting with a nucleic acid or antibody of thepresent invention.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent provided is attached to a solidsupport, the solid support can be chromatograph media, a test platehaving a plurality of wells, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides. Generally speaking, kits in accordancewith the present invention will include a suitable crystal protein,peptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

2.3 ELISAs and Immunoprecipitation

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating crystal protein antigen sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 hours, at temperatures preferably on theorder of about 25° to about 27° C. Following incubation, theantisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween®, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

The anti-crystal protein antibodies of the present invention areparticularly useful for the isolation of other crystal protein antigensby immunoprecipitation. Immunoprecipitation involves the separation ofthe target antigen component from a complex mixture, and is used todiscriminate or isolate minute amounts of protein. For the isolation ofmembrane proteins cells must be solubilized into detergent micelles.Nonionic salts are preferred, since other agents such as bile salts,precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.enzyme-substrate pairs.

2.4 Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-peptide antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

2.5 Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies. In particular, the invention concerns epitopic coresequences derived from Cry proteins or peptides.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a crystal protein or polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the crystal protein orpolypeptide will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art.

The identification of Cry inmmunodominant epitopes, and/or theirfunctional equivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, for example, Jameson andWolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 8 to about 20 amino acids in length,and more preferably about 8 to about 15 amino acids in length. It isproposed that shorter antigenic crystal protein-derived peptides willprovide advantages in certain circumstances, for example, in thepreparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to crystal proteins,and in particular Cry and Cry-related sequences. These epitopic coresequences are identified herein in particular aspects as hydrophilicregions of the particular polypeptide antigen. It is proposed that theseregions represent those which are most likely to promote T-cell orB-cell stimulation, and, hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on the crystal protein-directed antibodiesdisclosed herein. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) mayalso be useful in designing synthetic peptides in accordance with thepresent disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat about 4° C., or more preferably, frozen. Of course, where thepeptides are stored in a lyophilized or powdered state, they may bestored virtually indefinitely, e.g., in metered aliquots that may berehydrated with a predetermined amount of water (preferably distilled)or buffer prior to use.

2.6 Nucleic Acid Segments Encoding Crystal Protein Chimeras

The present invention also concerns DNA segments, both native,synthetic, and mutagenized, that can be synthesized, or isolated fromvirtually any source, that are free from total genomic DNA and thatencode the novel chimeric peptides disclosed herein. DNA segmentsencoding these peptide species may prove to encode proteins,polypeptides, subunits, functional domains, and the like of crystalprotein-related or other non-related gene products. In addition theseDNA segments may be synthesized entirely in vitro using methods that arewell-known to those of skill in the art.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a crystal protein or peptide refers toa DNA segment that contains crystal protein coding sequences yet isisolated away from, or purified free from, total genomic DNA of thespecies from which the. DNA segment is obtained, which in the instantcase is the genome of the Gram-positive bacterial genus, Bacillus, andin particular, the species of Bacillus known as B. thuringiensis.Included within the term “DNA segment”, are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified crystalprotein-encoding gene refers to a DNA segment which may include inaddition to peptide encoding sequences, certain other elements such as,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein-encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein-,polypeptide- or peptide-encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences, operonsequences and smaller engineered gene segments that express, or may beadapted to express, proteins, polypeptides or peptides.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a gene encoding a bacterial crystalprotein, forms the significant part of the coding region of the DNAsegment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or operon coding regions. Of course, this refersto the DNA segment as originally isolated, and does not exclude genes,recombinant genes, synthetic linkers, or coding regions later added tothe segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode a Crypeptide species that includes within its amino acid sequence an aminoacid sequence essentially as set forth in SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34.

The term “a sequence essentially as set forth in SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ IDNO:34” means that the sequence substantially corresponds to a portion ofthe sequence of either SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34 and has relativelyfew amino acids that are not identical to, or a biologically functionalequivalent of, the amino acids of any of these sequences. The term“biologically functional equivalent” is well understood in the art andis further defined in detail herein (e.g., see IllustrativeEmbodiments). Accordingly, sequences that have between about 70% andabout 80%, or more preferably between about 81% and about 90%, or evenmore preferably between about 91% and about 99% amino acid sequenceidentity or functional equivalence to the amino acids of SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, orSEQ ID NO:34 will be sequences that are “essentially as set forth in SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, or SEQ ID NO:34.”

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various noncoding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding either of the peptidesequences disclosed in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, or that areidentical to or complementary to DNA sequences which encode any of thepeptides disclosed in SEQ ID NO:10, SEQ ID NO:12 SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, and particularlythose DNA segments disclosed in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. For example,DNA sequences such as about 14 nucleotides, and that are up to about10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500,about 200, about 100, about 50, and about 14 base pairs in length(including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that “intermediate lengths”, in thesecontexts, means any length between the quoted ranges, such as 14, 15,16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51,52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through the 200-500; 500-1,000; 1,000-2,000;2,000-4,000; 3,000-5,000; and up to and including sequences of about10,000 nucleotides and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequences of SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQID NO:34, including those DNA sequences which are particularly disclosedin SEQ ID NO:9, SEQ ID NO:11 SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27,SEQ ID NO:29, or SEQ ID NO:33. Recombinant vectors and isolated DNAsegments may therefore variously include the peptide-coding regionsthemselves, coding regions bearing selected alterations or modificationsin the basic coding region, or they may encode larger polypeptides thatnevertheless include these peptide-coding regions or may encodebiologically functional equivalent proteins or peptides that havevariant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology; in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.7 Recombinant Vectors and Protein Expression

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or plant cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell, type, organism, or even animal, chosenfor expression. The use of promoter and cell type combinations forprotein expression is generally known to those of skill in the art ofmolecular biology, for example, see Sambrook et al, 1989. The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides. Appropriate promoter systemscontemplated for use in high-level expression include, but are notlimited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful. Such peptideepitopes may be amino acid sequences which comprise contiguous aminoacid sequences from SEQ ID NO:10, SEQ ID NO:12 SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34; or any peptideepitope encoded by the nucleic acid sequences of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:33.

Methods for the recombinant expression of crystal proteins and vectorsuseful in the expression of DNA constructs encoding crystal proteins aredescribed in Intl. Pat. Appl. Publ. No. WO 95/02058, specificallyincorporated herein by reference.

Recombinant Host Cells

TABLE 2 STRAINS DEPOSITED WITH NRRL STRAIN PLASMID ACCESSION NUMBERDEPOSIT DATE EG11063 pEG1068 B-21579 Jun. 26, 1996 EG11074 pEG1077B-21580 Jun. 26, 1996 EG11091 pEG1092 B-21780 May XX, 1997 EG11092pEG1093 B-21635 Nov. 14, 1996 EG11735 pEG365 B-21581 Jun. 26, 1996EG11751 pEG378 B-21636 Nov. 14, 1996 EG11768 pEG381 B-21781 May XX, 1997

DNA Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of crystal proteinsor peptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least a 14 nucleotidelong contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, or SEQ ID NO:33 will find particular utility. Also, nucleic acidsegments which encode at least a 6 amino acid contiguous sequence fromSEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, or SEQ ID NO:34, are also preferred. Longer contiguousidentical or complementary sequences, e.g., those of about 20, 30, 40,50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. (including allintermediate lengths and up to and including full-length sequences willalso be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize tocrystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200nucleotides or so, identical or complementary to DNA sequences of SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, or SEQ ID NO:33, are particularly contemplated as hybridizationprobes for use in, e.g., Southern and Northern blotting. Smallerfragments will generally find use in hybridization embodiments, whereinthe length of the contiguous complementary region may be varied, such asbetween about 10-14 and about 100 or 200 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCRT technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each specifically incorporated herein by reference), byintroducing selected sequences into recombinant vectors for recombinantproduction, and by other recombinant DNA techniques generally known tothose of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating crystalprotein-encoding DNA segments. Detection of DNA segments viahybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each specificallyincorporated herein by reference) are exemplary of the methods ofhybridization analyses. Teachings such as those found in the texts ofMaloy et al., 1994; Segal 1976; Prokop, 1991; and Kuby, 1994, areparticularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.10 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein, andconsequently increasing the insecticidal activity and/or expression ofthe recombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 3.

TABLE 3 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

2.11 Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

2.12 Crystal Protein Compositions as Insecticides and Methods of Use

The inventors contemplate that the chimeric crystal protein compositionsdisclosed herein will find particular utility as insecticides fortopical and/or systemic application to field crops, grasses, fruits andvegetables, and ornamental plants. In a preferred embodiment, thebioinsecticide composition comprises an oil flowable suspension ofbacterial cells which expresses a novel crystal protein disclosedherein. Preferably the cells are B. thuringiensis cells, however, anysuch bacterial host cell expressing the novel nucleic acid segmentsdisclosed herein and producing a crystal protein is contemplated to beuseful, such as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.

In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel crystal protein disclosed herein.Preferred bacterial cells are B. thuringiensis cells, however, bacteriasuch as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing thecrystal protein are also contemplated to be useful.

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, pellet, or collodial concentrate.This powder comprises bacterial cells which expresses a novel crystalprotein disclosed herein. Preferred bacterial cells are B. thuringiensiscells, however, bacteria such as B. megaterium, B. subtilis, E. coli, orPseudomonas spp. cells transformed with a DNA segment disclosed hereinand expressing the crystal protein are also contemplated to be useful.Such dry forms of the insecticidal compositions may be formulated todissolve immediately upon wetting, or alternatively, dissolve in acontrolled-release, sustained-release, or other time-dependent manner.

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above which express the crystal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solutionready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise intact B. thuringiensiscells expressing the protein of interest, such bacteria may beformulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (phyllosilicates, carbonates, sulfates,phosphates, and the like) or botanical materials (powdered corncobs,rice hulls, walnut shells, and the like). The formulations may includespreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel chimeric Cry proteins may be prepared byrecombinant bacterial expression systems in vitro and isolated forsubsequent field application. Such protein may be either in crude celllysates, suspensions, colloids, etc., or alternatively may be purified,refined, buffered, and/or further processed, before formulating in anactive biocidal formulation. Likewise, under certain circumstances, itmay be desirable to isolate crystals and/or spores from bacterialcultures expressing the crystal protein and apply solutions,suspensions, or collodial preparations of such crystals and/or spores asthe active bioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) are applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, dessicated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target coleopteran insect, typically onto the foliageof the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well-known to those ofskill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication; environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 0.5% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 0.5% to about 99% or more by weightof the composition, while liquid formulations may generally comprisefrom about 0.5% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 10⁴ to about 10¹² cells/mg.

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 50g to about 500 g of active ingredient, or of from about 500 g to about1000 g, or of from about 1000 g to about 5000 g or more of activeingredient.

2.13 Antibody Compositions and Methods for Producing

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the crystalproteins disclosed herein. Means for preparing and characterizingantibodies are well known in the art (See, e.g., Harlow and Lane, 1988;incorporated herein by reference). The methods for generating monoclonalantibodies (mAbs) generally begin along the same lines as those forpreparing polyclonal antibodies. Briefly, a polyclonal antibody isprepared by immunizing an animal with an immunogenic composition inaccordance with the present invention and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or agoat. Because of the relatively large blood volume of rabbits, a rabbitis a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265 (specificallyincorporated herein by reference). Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified crystal protein, polypeptide orpeptide. The immunizing composition is administered in a mannereffective to stimulate antibody producing cells. Rodents such as miceand rats are preferred animals, however, the use of rabbit, sheep frogcells is also possible. The use of rats may provide certain advantages(Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mousebeing most preferred as this is most routinely used and generally givesa higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1Ag 4 1, Sp210-Ag14, FO, NSOJU, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, L1CR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (vol./vol.) PEG, (Gefter et al., 1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A and 1B. The wild-type δ-endotoxins and the relevant restrictionsites that were used to construct the hybrid δ-endotoxins pertinent tothe invention are diagrammed in FIG. 1A. Only the DNA encoding theδ-endotoxin that is contained on the indicated plasmid (identified bythe “pEG” prefix) is shown. The B. thuringiensis strains containing theindicated plasmids are identified by the “EG” prefix. The hybridδ-endotoxins described in the invention are diagrammed in FIG. 1B andare aligned with the wild-type δ-endotoxins in FIG. 1A.

FIG. 2. An equal amount of each washed sporulated B. thuringiensisculture was analyzed by SDS-PAGE. Lane a: control Cry1Ac producing B.thuringiensis strain EG11070, b: EG11060, c: EG11062, d: EG11063, e:EG11065, F: EG11067, g: EG11071, h: EG11073,i: EG11074,j: EG11088,k:EG11090, and l: EG11091.

FIG. 3. Solubilized hybrid δ-endotoxins were exposed to trypsin for 0,15, 30, 60, and 120 minutes. The resulting material was analyzed bySDS-PAGE. The amount of active δ-endotoxin fragment remaining wasquantitated by scanning densitometry using a Molecular Dynamics model300A densitometer. The percent active toxin remaining was plotted versustime. Wild-type Cry1Ac δ-endotoxin (open box) served as the control.

FIG. 4. Schematic diagrams of the wild-type toxins and the relevantrestriction sites that were used to construct the hybrid δ-endotoxinencoded by pEG381 and expressed in EG11768. Only the DNA encoding theδ-endotoxin that is contained on the indicated plasmid (identified bythe “pEG” prefix) is shown.

4. BRIEF DESCRIPTION OF THE SEQUENCES

   SEQ ID NO:l is oligonucleotide primer A.    SEQ ID NO:2 isoligonucleotide primer B.    SEQ ID NO:3 is oligonucleotide primer C.   SEQ ID NO:4 is oligonucleotide primer D.    SEQ ID NO:5 isoligonucleotide primer E.    SEQ ID NO:6 is oligonucleotide primer F.   SEQ ID NO:7 is oligonucleotide primer G.    SEQ ID NO:8 isoligonucleotide primer H.

SEQ ID NO:9 is the nucleotide and deduced amino acid sequences of theEG11063 hybrid δ-endotoxin.

SEQ ID NO:10 denotes in the three-letter abbreviation form, the aminoacid sequence for the hybrid δ-endotoxin specified in SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide and deduced amino acid sequences of theEG11074 hybrid δ-endotoxin.

SEQ ID NO:12 denotes in the three-letter abbreviation form, the aminoacid sequence for the hybrid δ-endotoxin specified in SEQ ID NO:11.

SEQ ID NO:13 is the nucleotide and deduced amino acid sequences of theEG11735 hybrid δ-endotoxin.

SEQ ID NO:14 denotes in the three-letter abbreviation form, the aminoacid sequence for the hybrid δ-endotoxin specified in SEQ ID NO:13.

SEQ ID NO:15 is the 5′ exchange site for pEG065, pEG1070, and pEG1074.

SEQ ID NO:16 is the 5′ exchange site for pEG1067, pEG1072, and pEG1076.

SEQ ID NO:17 is the 5′ exchange site for pEG1068, pEG1077, and pEG365.

SEQ ID NO:18 is the 5′ exchange site for pEG1088 and pEG1092.

SEQ ID NO:19 is the 5′ exchange site for pEG1089 and the 3′ exchangesite for pEG1070 and pEG1072.

SEQ ID NO:20 is the 5′ exchange site for pEG1091.

SEQ ID NO:21 is the 3′ exchange site for pEG1065, pEG1067, pEG1068,pEG1093, pEG378, and pEG365.

SEQ ID NO:22 is the 3′ exchange site for pEG1088.

SEQ ID NO:23 is oligonucleotide Primer I.

SEQ ID NO:24 is oligonucleotide Primer J.

SEQ ID NO:25 is the nucleic acid sequence and deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11092.

SEQ ID NO:26 is the three-letter abbreviation form of the amino acidsequence of the hybrid crystal protein produced by strain EG11092encoded by SEQ ID NO:25.

SEQ ID NO:27 is the nucleic acid sequence and the deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11751.

SEQ ID NO:28 is the three-letter abbreviation form of the amino acidsequence of the hybrid crystal protein produced by strain EG11751encoded by SEQ ID NO:27.

SEQ ID NO:29 is the nucleic acid sequence and the deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11091.

SEQ ID NO:30 is the three-letter abbreviation form of the amino acidsequence of the hybrid crystal protein produced by strain EG11091encoded by SEQ ID NO:29.

SEQ ID NO:31 is oligonucleotide primer K.

SEQ ID NO:32 is the 5′ exchange site for pEG378 and pEG381.

SEQ ID NO:33 is the nucleic acid sequence and the deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11768.

SEQ ID NO:34 denotes in the three-letter abbreviation form, the aminoacid sequence of the hybrid crystal protein produced by strain EG11768encoded by SEQ ID NO:33.

SEQ ID NO:35 is the 3′ exchange site for pEG1074, pEG1076, pEG1077 andpEG381.

5. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

5.1 Methods for Culturing B. Thuringiensis to Produce Cry Proteins

The B. thuringiensis strains described herein may be cultured usingstandard known media and fermentation techniques. Upon completion of thefermentation cycle, the bacteria may be harvested by first separatingthe B. thuringiensis spores and crystals from the fermentation broth bymeans well known in the art. The recovered B. thuringiensis spores andcrystals can be formulated into a wettable powder, a liquid concentrate,granules or other formulations by the addition of surfactants,dispersants, inert carriers and other components to facilitate handlingand application for particular target pests. The formulation andapplication procedures are all well known in the art and are used withcommercial strains of B. thuringiensis (HD-1) active againstLepidoptera, e.g., caterpillars.

5.2 Recombinant Host Cells for Expression of Cry Genes

The nucleotide sequences of the subject invention can be introduced intoa wide variety of microbial hosts. Expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the sites of lepidopteran insects where they willproliferate and be ingested by the insects. The results is a control ofthe unwanted insects. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinproduced in the cell. The treated cell then can be applied to theenvironment of target pest(s). The resulting product retains thetoxicity of the B. thuringiensis toxin.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility or toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and Gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B. thuringiensisgene into the host, availability of expression systems, efficiency ofexpression, stability of the pesticide in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apesticide microcapsule include protective qualities for the pesticide,such as thick cell walls, pigmentation, and intracellular packaging orformation of inclusion bodies; leaf affinity; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the toxin; and the like. Other considerationsinclude ease of formulation and handling, economics, storage stability,and the like.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.Specific organisms include Pseudomonas aeruginosa, P. fluorescens,Saccharomyces cerevisiae, B. thuringiensis, B. subtilis, E. coli,Streptomyces lividans and the like.

Treatment of the microbial cell, e.g., a microbe containing the B.thuringiensis toxin gene, can be by chemical or physical means, or by acombination of chemical and/or physical means, so long as the techniquedoes not deleteriously affect the properties of the toxin, nor diminishthe cellular capability in protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as formaldehyde andglutaraldehye; anti-infectives, such as zephiran chloride andcetylpyridinium chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Lugol's iodine, Bouin's fixative,and Helly's fixatives, (see e.g., Humason, 1967); or a combination ofphysical (heat) and chemical agents that preserve and prolong theactivity of the toxin produced in the cell when the cell is administeredto a suitable host. Examples of physical means are short wavelengthradiation such as γ-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. The cells employed will usually be intactand be substantially in the proliferative form when treated, rather thanin a spore form, although in some instances spores may be employed.

Where the B. thuringiensis toxin gene is introduced via a suitablevector into a microbial host, and said host is applied to theenvironment in a living state, it is essential that certain hostmicrobes be used. Microorganism hosts are selected which are known tooccupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment (crop and other insect habitats) with the wild-typemicroorganisms, provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Bacillus,Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kliqveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes eutrophus, andAzotobacter vinlandii; and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. auraniaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans.

5.3 Definitions

The following words and phrases have the meanings set forth below.

Broad-Spectrum: refers to a wide range of insect species.

Broad-Spectrum Insecticidal Activity: toxicity towards a wide range ofinsect species.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Insecticidal Activity: toxicity towards insects.

Insecticidal Specificity: the toxicity exhibited by a crystal proteintowards multiple insect species.

Intraorder Specificity: the toxicity of a particular crystal proteintowards insect species within an Order of insects (e.g., OrderLepidoptera).

Interorder Specificity: the toxicity of a particular crystal proteintowards insect species of different Orders (e.g., Orders Lepidoptera andDiptera).

LC₅₀: the lethal concentration of crystal protein that causes 50%mortality of the insects treated.

LC₉₅: the lethal concentration of crystal protein that causes 95%mortality of the insects treated.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast or explant).

Structural Gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication.

Transformed Cell: A cell whose DNA has been altered by the introductionof an exogenous DNA molecule into that cell.

Transgene: An exogenous gene which when introduced into the genome of ahost cell through a process such as transformation, electroporation,particle bombardment, and the like, is expressed by the host cell andintegrated into the cells genome such that the trait or traits producedby the expression of the transgene is inherited by the progeny of thetransformed cell.

Transgenic Cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell. Exemplary transgenic cells includeplant calli derived from a transformed plant cell and particular cellssuch as leaf, root, stem, e.g., somatic cells, or reproductive (germ)cells obtained from a transgenic plant.

Transgenic Plant: A plant or progeny thereof derived from a transformedplant cell or protoplast, wherein the plant DNA contains an introducedexogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callus obtained from a transformed plant cell orprotoplast as being a transgenic plant, and that usage will be followedherein.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

5.4 Probes and Primers

In another aspect, DNA sequence information provided by the inventionallows for the preparation of relatively short DNA (or RNA) sequenceshaving the ability to specifically hybridize to gene sequences of theselected polynucleotides disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of a selected crystal protein gene sequence, e.g., asequence such as that shown in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. The abilityof such nucleic acid probes to specifically hybridize to a crystalprotein-encoding gene sequence lends them particular utility in avariety of embodiments. Most importantly, the probes may be used in avariety of assays for detecting the presence of complementary sequencesin a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingPCR™ technology. Segments of related crystal protein genes from otherspecies may also be amplified by PCRT using such primers.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes sequences that are complementary to at leasta 14 to 30 or so long nucleotide stretch of a crystal protein-encodingsequence, such as that shown in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. A size of atleast 14 nucleotides in length helps to ensure that the fragment will beof sufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 14 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 14 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. Nos. 4,683,195, and 4,683,202 (each specifically incorporatedherein by reference), or by excising selected DNA fragments fromrecombinant plasmids containing appropriate inserts and suitablerestriction sites.

5.5 Expression Vectors

The present invention contemplates an expression vector comprising apolynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to an coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region.

As used herein, the term “operatively linked” means that a promoter isconnected to an coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region are well known inthe art.

Promoters that function in bacteria are well known in the art. Exemplaryand preferred promoters for the Bacillus crystal proteins include thesigA, sigE, and sigK gene promoters. Alternatively, the native,mutagenized, or recombinant crystal protein-encoding gene promotersthemselves can be used.

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in plants. Promoters that function in plants are also wellknown in the art. Useful in expressing the polypeptide in plants arepromoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

A promoter is also selected for its ability to direct the transformedplant cell's or transgenic plant's transcriptional activity to thecoding region. Structural genes can be driven by a variety of promotersin plant tissues. Promoters can be near-constitutive, such as the CaMV35S promoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S,increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callus, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which isspecific for seed tissue. The Lectin protein in soybean seeds is encodedby a single gene (Le1) that is only expressed during seed maturation andaccounts for about 2 to about 5% of total seed mRNA. The lectin gene andseed-specific promoter have been fully characterized and used to directseed specific expression in transgenic tobacco plants (Vodkin et al.,1983; Lindstrom et al., 1990.)

An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yanget al., 1990), corn alcohol dehydrogenase I (Vogel et al., 1989), cornlight harvesting complex (Simpson, 1986), corn heat shock protein (Odellet al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986;Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein I(Keller et al., 1989), CaMV 35s transcript (Odell et al., 1985) andPotato patatin (Wenzler et al., 1989). Preferred promoters are thecauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunitRuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al.,1987). However, several other plant integrating vector systems are knownto function in plants including pCaMVCN transfer control vectordescribed (Fromm et al., 1985). pCaMVCN (available from Pharmacia,Piscataway, N.J.) includes the cauliflower mosaic virus CaMV 35Spromoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3N non-translated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011 (each of which is specificallyincorporated herein by reference). Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

A variety of methods has been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to conferinsecticidal activity to a cell is preferably a chimeric B.thuringiensis crystal protein-encoding gene. In preferred embodiments,such a polypeptide has the amino acid residue sequence of SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, orSEQ ID NO:34; or a functional equivalent of one or more of thosesequences. In accordance with such embodiments, a coding regioncomprising the DNA sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33 is alsopreferred.

5.6 Transformed or Transgenic Plant Cells

A bacterium, a yeast cell, or a plant cell or a plant transformed withan expression vector of the present invention is also contemplated. Atransgenic bacterium, yeast cell, plant cell or plant derived from sucha transformed or transgenic cell is also contemplated. Means fortransforming bacteria and yeast cells are well known in the art.Typically, means of transformation are similar to those well known meansused to transform other bacteria or yeast such as E. coli or S.cerevisiae.

Methods for DNA transformation of plant cells includeAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos and particle bombardment. Each of these methodshas distinct advantages and disadvantages. Thus, one particular methodof introducing genes into a particular plant strain may not necessarilybe the most effective for another plant strain, but it is well knownwhich methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as infection by A. tumefaciensand related Agrobacterium, direct delivery of DNA such as, for example,by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993),by desiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973);(2) physical methods such as microinjection (Capecchi, 1980),electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and thegene gun (Johnston and Tang, 1994; Fynan et at, 1993); (3) viral vectors(Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a; 1988b); and(4) receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner etal., 1992).

5.6.1 Electroporation

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of clones genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known tothose of skill in the art. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

5.6.2 Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with corncells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducingdamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

The methods of particle-mediated transformation is well-known to thoseof skill in the art. U.S. Pat. No. 5,015,580 (specifically incorporatedherein by reference) describes the transformation of soybeans using sucha technique.

5.6.3 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). The genetic engineering of cottonplants using Agrobacterium-mediated transfer is described in U.S. Pat.No. 5,004,863 (specifically incorporated herein by reference), while thetransformation of lettuce plants is described in U.S. Pat. No. 5,349,124(specifically incorporated herein by reference). Further, theintegration of the Ti-DNA is a relatively precise process resulting infew rearrangements. The region of DNA to be transferred is defined bythe border sequences, and intervening DNA is usually inserted into theplant genome as described (Spielmann et al., 1986; Jorgensen et al.,1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al, 1987). Therefore, commercially important cereal grains such asrice, corn, and wheat must usually be transformed using alternativemethods. However, as mentioned above, the transformation of asparagususing Agrobacterium can also be achieved (see, e.g., Bytebier et al.,1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

More preferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for enhanced carboxylase activity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (see, e.g., Fujimura et al., 1985; Toriyama et al., 1986;Yamada et al., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1988). Inaddition, “particle gun” or high-velocity microprojectile technology canbe utilized (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

5.7 Production of Insect-Resistant Transgenic Plants

Thus, the amount of a gene coding for a polypeptide of interest (i.e., abacterial crystal protein or polypeptide having insecticidal activityagainst one or more insect species) can be increased in plant such ascorn by transforming those plants using particle bombardment methods(Maddock et al, 1991). By way of example, an expression vectorcontaining a coding region for a B. thuringiensis crystal protein and anappropriate selectable marker is transformed into a suspension ofembryonic maize (corn) cells using a particle gun to deliver the DNAcoated on microprojectiles. Transgenic plants are regenerated fromtransformed embryonic calli that express the disclosed insecticidalcrystal proteins. Particle bombardment has been used to successfullytransform wheat (Vasil et al., 1992).

DNA can also be introduced into plants by direct DNA transfer intopollen as described (Zhou et al., 1983; Hess, 1987; Luo et al., 1988).Expression of polypeptide coding genes can be obtained by injection ofthe DNA into reproductive organs of a plant as described (Pena et al.,1987). DNA can also be injected directly into the cells of immatureembryos and the rehydration of desiccated embryos as described (Neuhauset al., 1987; Benbrook et al., 1986).

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art (Weissbach andWeissbach, 1988). This regeneration and growth process typicallyincludes the steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 (specification incorporated herein by reference) detailsthe creation of genetically transformed lettuce cells and plantsresulting therefrom which express hybrid crystal proteins conferringinsecticidal activity against Lepidopteran larvae to such plants.

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

A transgenic plant of this invention thus has an increased amount of acoding region (e.g., a cry gene) that encodes one or more of theChimeric Cry polypeptides disclosed herein. A preferred transgenic plantis an independent segregant and can transmit that gene and its activityto its progeny. A more preferred transgenic plant is homozygous for thatgene, and transmits that gene to all of its offspring on sexual mating.Seed from a transgenic plant may be grown in the field or greenhouse,and resulting sexually mature transgenic plants are self-pollinated togenerate true breeding plants. The progeny from these plants become truebreeding lines that are evaluated for, by way of example, increasedinsecticidal capacity against Coleopteran insects, preferably in thefield, under a range of environmental conditions. The inventorscontemplate that the present invention will find particular utility inthe creation of transgenic corn, soybeans, cotton, wheat, oats, barley,other grains, vegetables, fruits, fruit trees, berries, turf grass,ornamentals, shrubs and trees.

6. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

6.1 Example 1—Construction of Hybrid B. thuringiensis δ-Endotoxins

The B. thuringiensis shuttle vectors pEG853, pEG854, and pEG857 whichare used in the present invention have been described (Baum et al.,1990). pEG857 contains the Cry1Ac gene cloned into pEG853 as anSphI-BamHI DNA fragment. pEG1064 was constructed in such a way that theKpnI site within the cry1Ac gene was preserved and the KpnI site in thepEG857 multiple cloning site (MCS) was eliminated. This was accomplishedby sequentially subjecting pEG857 DNA to limited KpnI digestion so thatonly one KpnI site is cut, filling in the KpnI 5′ overhang by Klenowfragment of DNA polymerase I to create blunt DNA ends, and joining theblunt ends of DNA by T4 DNA ligase. pEG318 contains the cry1F gene(Chambers et al., 1991) cloned into the XhoI site of pEG854 as anXhoISalI DNA fragment. pEG315 contains the cry1C gene from strain EG6346(Chambers et al., 1991) cloned into the XhoI-BamHI sites of pEG854 as aSalI-BamHI DNA fragment.

FIG. 1A shows a schematic representation of the DNA encoding thecomplete cry1Ac, cry1Ab, cry1C, and cry1F genes contained onpEG854/pEG1064, pEG20, pEG315, and pEG318, respectively. Uniquerestriction sites that were used in constructing certain hybrid genesare also shown. FIG. 1B shows a schematic representation of hybrid genespertaining to the present invention. In some cases standard PCR™amplification with mutagenic oligonucleotide primers were used toincorporate appropriate restrictions sites into DNA fragments used forhybrid gene construction. Certain hybrid gene constructions could not beaccomplished by restriction fragment subcloning. In those instances,PCR™ overlap extension (POE) was used to construct the desired hybridgene (Horton et al., 1989). The following oligonucleotide primers(purchased from Integrated DNA Technologies Inc., Coralville, Iowa) wereused:

Primer A: 5′-GGATAGCACTCATCAAAGGTACC-3′ (SEQ ID NO:1) Primer B:5′-GAAGATATCCAATTCGAACAGTTTCCC-3′ (SEQ ID NO:2) Primer C:5′-CATATTCTGCCTCGAGTGTTGCAGTAAC-3′ (SEQ ID NO:3) Primer D:5′-CCCGATCGGCCGCATGC-3′ (SEQ ID NO:4) Primer E: 5′-CATTGGAGCTCTCCATG-3′(SEQ ID NO:5) Primer F: 5′-GCACTACGATGTATCC-3′ (SEQ ID NO:6) Primer G:5′-CATCGTAGTGCAACTCTTAC-3′ (SEQ ID NQ:7) Primer H:5′-CCAAGAAAATACTAGAGCTCTTGTTAAAAAAGGTGTTCC-3′ (SEQ ID NO:8) Primer I:5′-ATTTGAGTAATACTATCC-3′ (SEQ ID NO:23) Primer J:5′-ATTACTCAAATACCATTGG-3′ (SEQ ID NO:24) Primer K:5′-TCGTTGCTCTGTTCCCG-3′ (SEQ ID NO:31)

The plasmids described in FIG. 1B containing the hybrid δ-endotoxingenes pertinent to this invention are described below. Isolation orpurification of DNA fragments generated by restriction of plasmid DNA,PCR™ amplification, or POE refers to the sequential application ofagarose-TAE gel electrophoresis and use of the Geneclean Kit (Bio 101)following the manufacturer's recommendation. pEG1065 was constructed byPCR™ amplification of the cry1F DNA fragment using primer pair A and Band pEG318 as the DNA template. The resulting PCR™ product was isolated,cut with AsuII and KpnI, and used to replace the correspondingAsuII-KpnI DNA fragment in pEG857. Plasmid pEG1067 was constructed usingPOE and DNA fragments SauI-KpnI of cry1F and AsuII-ClaI of cry1Ac thatwere isolated from pEG318 and pEG857, respectively. The resulting POEproduct was PCR™ amplified with primer pair A and B, cut with AsuII andKpnI, and used to replace the corresponding AsuII-KpnI fragment inpEG857.

pEG1068 was constructed by replacing the SacI-KpnI DNA fragment ofcry1Ac isolated from pEG857 with the corresponding SacI-KpnI DNAfragment isolated from cry1F (pEG318). pEG1070 was constructed byreplacing the SacI-KpnI DNA fragment isolated from pEG1065 with thecorresponding SacI-KpnI DNA fragment isolated from cry1Ac (pEG857).pEG1072 was constructed by replacing the SacI-KpnI DNA fragment isolatedfrom pEG1067 with the corresponding SacI-KpnI DNA fragment isolated fromcry1Ac (pEG857). pEG1074, pEG1076, and pEG1077 were constructed byreplacing the SphI-XhoI DNA fragment from pEG1064 with the PCRTamplified SphI-XhoI DNA fragment from pEG1065, pEG1067, pEG1068,respectively, using primer pairs C and D. pEG1089 was constructed byreplacing the SphI-SacI DNA fragment of pEG1064 with the isolated andSphI and SacI cut PCR™ product of cry1F that was generated using primerpair D and E and the template pEG318.

pEG1091 was constructed by replacing the SphI-SacI DNA fragment ofpEG1064 with the isolated and SphI and SacI cut PCRT product of cry1Cthat was generated using primer pair D and H and the template pEG315.

pEG1088 was constructed by POE using a cry1Ac DNA fragment generatedusing primer pair B and F and a cry1C DNA fragment generated usingprimer pair A and G. The SacI-KpnI fragment was isolated from theresulting POE product and used to replace the corresponding SacI-KpnIfragment in pEG1064.

pEG365 was constructed by first replacing the SphI-KpnI DNA fragmentfrom pEG1065 with the corresponding cry1Ab DNA fragment isolated frompEG20 to give pEG364. The SacI-KpnI DNA fragment from pEG364 was thenreplaced with the corresponding cry1F DNA fragment isolated from pEG318.

pEG1092 was constructed by replacing the KpnI-BamHI DNA fragment frompEG1088 with the corresponding DNA fragment isolated from pEG315.pEG1092 is distinct from the cry1Ab/cry1C hybrid δ-endotoxin genedisclosed in Intl. Pat. Appl. Publ. No. WO 95/06730.

pEG1093 was constructed by replacing the SphI-AsuII DNA fragment frompEG1068 with the corresponding SphI-AsuII DNA fragment isolated frompEG20.

pEG378 was constructed by POE using a cry1Ac DNA fragment generatedusing primer pair B and I using pEG857 as the template and a cry1F DNAfragment generated using primer pair A and J using pEG318 as thetemplate. The resulting POE product was cut with AsuII and KpnI and theresulting isolated DNA fragment used to replace the correspondingAsuII-KpnI DNA fragment in pEG1064.

pEG381 was constructed by replacing the AsuII-XhoI DNA fragment inpEG1064 with the corresponding AsuII-XhoI DNA fragment isolated from thePCR™ amplification of pEG378 using primer pair C and K.

6.2 Example 2—Production of the Hybrid Toxins in B. Thuringiensis

The plasmids encoding the hybrid toxins described in Example 1 weretransformed into B. thuringiensis as described (Mettus and Macaluso,1990). The resulting B. thuringiensis strains were grown in 50 ml of C-2medium until the culture was fully sporulated and lysed (approximately48 hr.). Since crystal formation is a prerequisite for efficientcommercial production of δ-endotoxins in B. thuringiensis, microscopicanalysis was used to identify crystals in the sporulated cultures (Table4).

TABLE 4 CRYSTAL FORMATION BY THE HYBRID δ-ENDOTOXINS Crystal StrainPlasmid Parent δ-Endotoxins Formation EG11060 pEG1065 Cry1Ac + Cry1F +EG11062 pEG1067 Cry1Ac + Cry1F + EG11063 pEG1068 Cry1Ac + Cry1F +EG11065 pEG1070 Cry1Ac + Cry1F − EG11067 pEG1072 Cry1Ac + Cry1F −EG11071 pEG1074 Cry1Ac + Cry1F + EG11073 pEG1076 Cry1Ac + Cry1F +EG11074 pEG1077 Cry1Ac + Cry1F + EG11087 pEG1088 Cry1Ac + Cry1C −EG11088 pEG1089 Cry1F + Cry1Ac − EG11090 pEG1091 Cry1C + Cry1Ac −EG11091 pEG1092 Cry1Ac + Cry1C + EG11092 pEG1093 Cry1Ab + Cry1Ac +Cry1F + EG11735 pEG365 Cry1Ab + Cry1F + Cry1Ac + EG11751 pEG378 Cry1Ac +Cry1F + EG11768 pEG381 Cry1Ac + Cry1F +

The δ-endotoxin production for some of the B. thuringiensis strainsspecified in Table 4 was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described byBaum et al., 1990. Equal volume cultures of each B. thuringiensis strainwere grown in C-2 medium until fully sporulated and lysed. The cultureswere centrifuged and the spore/crystal pellet was washed twice withequal volumes of distilled deionized water. The final pellet wassuspended in half the culture volume of 0.005% Triton X-100®. An equalvolume of each washed culture was analyzed by SDS-PAGE as shown in FIG.2.

The majority of hybrids involving Cry1Ac and Cry1F formed stablecrystals in B. thuringiensis A notable exception is EG11088 in which theactive toxin fragment would be the reciprocal exchange of EG11063. Twoof the three hybrids involving Cry1Ac and Cry1C, EG11087 and EG11090,Failed to produce crystal in B. thuringiensis even though thesereciprocal hybrids mimic the activated toxin fragments ofcrystal-forming EG11063 and EG11074.

Every strain that was examined by SDS-PAGE produced some level ofδ-endotoxin. As expected, however, those cultures identified as crystalnegative produced very little protein (e.g., lane e: EG11065, lane f:EG11067, lane j: EG11088, and lane k: EG11090). For reference, typicalyields from a crystal forming δ-endotoxin is shown for Cry1Ac (lane a).Several hybrid δ-endotoxins produce comparable levels of proteinincluding EG11060 (lane b), EG11062 (lane c), EG11063 (lane d; SEQ IDNO:10), and EG11074 (lane i; SEQ ID NO:12). The data clearly show thatefficient hybrid δ-endotoxin production in B. thuringiensis isunpredictable and varies depending on the parent δ-endotoxins used toconstruct the hybrid.

6.3 Example 3—Proteolytic Processing of the Hybrid δ-Endotoxins

Proteolytic degradation of the protoxin form of the δ-endotoxin to astable active toxin occurs once δ-endotoxin crystals are solubilized inthe larval midgut. One measure of the potential activity of δ-endotoxinsis the stability of the active δ-endotoxin in a proteolytic environment.To test the proteolytic sensitivity of the hybrid δ-endotoxins,solubilized toxin was subjected to trypsin digestion. The δ-endotoxinswere purified from sporulated B. thuringiensis cultures and quantifiedas described (Chambers et al., 1991). Exactly 250 μg of each hybridδ-endotoxin crystal was solubilized in 30 mM NaHCO₃, 10 mM DTT (totalvolume 0.5 ml). Trypsin was added to the solubilized toxin at a 1:10ratio. At appropriate time points 50 μl aliquots were removed to 50 μlLaemmli buffer, heated to 100° C. for 3 min., and frozen in a dry-iceethanol bath for subsequent analysis. The trypsin digests of thesolubilized toxins were analyzed by SDS-PAGE and the amount of activeδ-endotoxin at each time point was quantified by densitometry. A graphicrepresentation of the results from these studies are shown in FIG. 3.

The wild-type Cry1Ac is rapidly processed to the active δ-endotoxinfragment that is stable for the duration of the study. The hybridδ-endotoxins from EG11063 and EG11074 are also processed to activeδ-endotoxin fragments which are stable for the duration of the study.The processing of the EG11063 δ-endotoxin occurs at a slower rate and ahigher percentage of this active δ-endotoxin fragment remains at eachtime point. Although the hybrid δ-endotoxins from EG11060 and EG11062are process to active δ-endotoxin fragments, these fragments are moresusceptible to further cleavage and degrade at various rates during thecourse of the study. The 5′ exchange points between cry1Ac and cry1F forthe EG11062 and EG11063 δ-endotoxins result in toxins that differ byonly 21 amino acid residues (see FIG. 1). However, the importance ofmaintaining Cry1Ac sequences at these positions is evident by the morerapid degradation of the EG11062 δ-endotoxin. These data demonstratethat different hybrid δ-endotoxins constructed using the same parentalδ-endotoxins can vary significantly in biochemical characteristics suchas proteolytic stability.

6.4 Example 4—Bioactivity of the Hybrid δ-Endotoxins

B. thuringiensis cultures expressing the desired δ-endotoxin were grownuntil fully sporulated and lysed and washed as described in Example 2.The δ-endotoxin levels for each culture were quantified by SDS-PAGE asdescribed (Baum et al., 1990). In the case of bioassay screens, a singleappropriate concentration of each washed δ-endotoxin culture wastopically applied to 32 wells containing 1.0 ml artificial diet per well(surface area of 175 m²). A single neonate larvae was placed in each ofthe treated wells and the tray covered by a clear perforated mylarsheet. Larvae mortality was scored after 7 days of feeding and percentmortality expressed as the ratio of the number of dead larvae to thetotal number of larvae treated, 32.

In the case of LC₅₀ determinations (δ-endotoxin concentration giving 50%mortality), δ-endotoxins were purified from the B. thuringiensiscultures and quantified as described by Chambers et al. (1991). Eightconcentrations of the δ-endotoxins were prepared by serial dilution in0.005% Triton X-100® and each concentration was topically applied towells containing 1.0 ml of artificial diet. Larvae mortality was scoredafter 7 days of feeding (32 larvae for each δ-endotoxin concentration),In all cases the diluent served as the control.

A comparison of the Cry1A/Cry1F hybrid toxins by bioassay screens isshown in Table 5. The hybrid δ-endotoxins from strains EG11063 andEG11074 maintain the activities of the parental Cry1Ac and Cry1Fδ-endotoxins. Furthermore, the hybrid δ-endotoxin from EG11735 maintainsthe activity of its parental Cry1Ab and Cry1F δ-endotoxins. Theδ-endotoxins produce by strains EG11061, EG11062, EG11071, and EG11073have no insecticidal activity on the insect larvae tested despite 1)being comprised of at least one parental δ-endotoxin that is activeagainst the indicated larvae and 2) forming stable, well-definedcrystals in B. thuringiensis. These results demonstrate theunpredictable nature of hybrid toxin constructions.

For the data in Table 5. All strains were tested as washed sporulatedcultures. For each insect tested, equivalent amounts of δ-endotoxinswere used and insecticidal activity was based on the strain showing thehighest percent mortality (++++).

TABLE 5 BIOASSAY SCREENS OF HYBRID CRY1A/CRY1F δ-ENDOTOXINS S. Strainfrugiperda S. exigua H. virescens H. zea O. nubilalis Cry1Ac − − ++++++++ +++ Cry1F ++++ ++ ++ ++ ++ Cry1Ab ++ + +++ ++ +++ EG11060 − − − − −EG11062 − − − − − EG11063 ++++ ++++ +++ +++ ++++ EG11071 − − − − −EG11073 − − − − − EG11074 ++++ ++++ +++ +++ ++++ EG11090 − +++ − − −EG11091 ++++ ++++ − − N.D. EG11092 ++++ ++++ +++ +++ N.D. EG11735 ++++++++ +++ +++ N.D. EG11751 N.D.^(a) ++++ N.D. ++++ N.D. ^(a)N.D. = notdetermined.

The δ-endotoxins described in FIG. 1 and that demonstrated insecticidalactivity in bioassay screens were tested as purified crystals todetermine their LC₅₀ (see Table 6). The δ-endotoxins purified fromstrains EG11063, EG11074, EG11091, and EG11735 all show increasedarmyworm (S. frugiperda and S. exigua) activity compared to any of thewild-type δ-endotoxins tested. The EG11063 and EG11074 δ-endotoxinswould yield identical active toxin fragments (FIG. 1B) which is evidentby their similar LC50 values on the insects examined. An unexpectedresult evident from these data is that a hybrid δ-endotoxin such asEG11063, EG11092, EG11074, EG11735, or EG11751 can retain the activityof their respective parental δ-endotoxins, and, against certain insectssuch as S. exigua, can have activity far better than either parentalδ-endotoxin. This broad range of insecticidal activity at doses close toor lower than the parental δ-endotoxins, along with the wild-type levelof toxin production (Example 2), make these proteins particularlysuitable for production in B. thuringiensis. Although the EG11091derived δ-endotoxin has better activity against S. frugiperda and S.exigua than its parental δ-endotoxins, it has lost the H. virescens andH. zea activity attributable to its Cry1Ac parent. This restricted hostrange along with lower toxin yield observed for the EG11091 δ-endotoxin(Example 2) make it less amenable to production in B. thuringiensis.

TABLE 6 LC₅₀ VALUES FOR THE PURIFIED HYBRID δ-ENDOTOXIN^(A) S. H. Toxinfrugiperda S. exigua virescens H. zea O. nubilalis Cry1Ac >10000 >100009 100 23 Cry1Ab 1435 4740 118 400 17 Cry1C >10000490 >10000 >10000 >10000 Cry1F 1027 3233 54 800 51 EG11063 550 114 33 807 (Cry1Ac/1F) EG11074 468 77 25 76 9 (Cry1Ac/1F) EG11091 21 21219 >10000 N.D.^(a) (Cry1Ac/1C) ^(a)N.D. = not determined.

In Table 6, the LC₅₀ values are expressed in nanograms of purifiedδ-endotoxin per well (175 mm²) and are the composite values for 2 to 6replications. nd=not determined.

TABLE 7 DNA EXCHANGE SITES FOR CRY1 HYBRID δ-ENDOTOXINS Plasmid SEQ IDNO: 5′ Exchange Site SEQ ID NO: 3′ Exchange Site pEG1065 15TATCCAATTCGAACGTCATC 21 ACTACCAGGTACCTTTGATG pEG1067 16TTTAGTCATCGATTAAATCA 21 ACTACCAGGTACCTTTGATG pEG1068 17ATAATAAGAGCTCCAATGTT 21 ACTACCAGGTACCTTTGATG pEG1070 15TATCCAATTCGAACGTCATC 19 TCATGGAGAGCTCCTATGTT pEG1072 16TTTAGTCATCGATTAAATCA 19 TCATGGAGAGCTCCTATGTT pEG1074 15TATCCAATTCGAACGTCATC 35 TGCAACACTCGAGGCTGAAT pEG1076 16TTTAGTCATCGATTAAATCA 35 TGCAACACTCGAGGCTGAAT pEG1077 17ATAATAAGAGCTCCAATGTT 35 TGCAACACTCGAGGCTGAAT pEG1088 18TACATCGTAGTGCAACTCTT 22 ACTACCGGGTACCTTTGATA pEG1089 19TCATGGAGAGCTCCTATGTT — NA pEG1091 20 TTAACAAGAGCTCCTATGTT — NA pEG109218 TACATCGTAGTGCAACTCTT — NA pEG1093 — ND^(b) 21 ACTACCAGGTACCTTTGATGpEG365 17 ATAATAAGAGCTCCAATGTT 21 ACTACCAGGTACCTTTGATG pEG378 32TCAAATACCATTGGTAAAAG 21 ACTACCAGGTACCTTTGATG pEG381 32TCAAATACCATTGGTAAAAG 35 TGCAACACTCGAGGCTGAAT ^(a)NA = Not Applicable.These hybrid toxins contain only one exchange site as shown in FIG. 1.^(b)ND = Not Distinguishable. The exchange site for these hybridproteins are identified by DNA sequences that are not distinguishablefrom either of the parent toxins.

Table 7 describes the DNA surrounding the 5′ and 3′ exchange points forthe hybrid δ-endotoxins which are pertinent to the present invention. Asevident by the SEQ ID NO, certain hybrid δ-endotoxins share exchangesites.

To examine the effect of other small changes in the exchange site chosenfor hybrid endotoxin construction, the activity of EG11751 and EG11063on S. exigua and H. zea were compared (Table 8). The data clearly showthat hybrid δ-endotoxin improvements can be made by altering theexchange site between the two parental δ-endotoxins. In this example,the exchange site in the EG11751 δ-endotoxin was moved 75 base pairs 3′compared to the EG11063 δ-indotoxin and results in improved insecticidalactivity. Although no significant improvement in S. exigua activity isobserved between EG11063 and EG11751, a significant improvement in H.zea activity of almost 4-fold is observed for EG11751. It is importantto note that improvements in hybrid δ-endotoxin bioactivity by alteringexchange sites is unpredictable. In the case of EG11062, moving theexchange site 63 base pairs 5′ of the EG11063 exchange site abolishesinsecticidal activity as shown in Table 7.

TABLE 8 BIOACTIVITY OF EG11063 AND EG11751 LC₅₀ Values for WashedSporulated Cultures B. thuringiensis Strain S. exigua H. zea EG11063 10638 EG11751  90 10

To further examine the effect of changes in the exchange site for hybridδ-endotoxins, the hybrid δ-endotoxin encoded by pEG381 was compared tothose encoded by pEG378 and pEG1068. In this example, the 3′ exchangesite for the pEG381 encoded hybrid δ-endotoxin was moved 340 base pairs5′ compared to the pEG378 hybrid δ-endotoxin. The data in Table 8 showthat this change results in an increase in S. frugiperda activitycompared to the pEG378 and pEG1066 encoded δ-endotoxins whilemaintaining the increased activity that was observed for the pEG378encoded δ-endotoxin over the pEG1068 encoded δ-endotoxin (see Table 7).This result is unexpected since the activated toxin resulting from theproteolysis of the encoded δ-endotoxins from pEG378 and pEG381 should beidentical. This example further demonstrates that exchange sites withinthe protoxin fragment of δ-endotoxins can have a profound effect oninsecticidal activity.

TABLE 9 BIOACTIVITY OF TOXINS ENCODED BY pEG378, pEG381 AND pEG1068 LC₅₀Values for Purified Crystals Plasmid S. frugiperda T. ni H. zea P.xylostella pEG378 464 57.7 37.5 3.02 pEG381 274 56.0 36.6 2.03 pEG1068476 66.7 72.7 3.83

6.5 Example 5—Activity of the Hybrid Toxins on Additional Pests

The toxins of the present invention were also assayed against additionalpests, including the southwestern corn borer and two pests activeagainst soybean. Toxin proteins were solubilized, added to diet andbioassayed against target pests. The hybrid toxins showed very effectivecontrol of all three pests.

TABLE 10 LC₅₀ AND EC₅₀ RANGES OF HYBRID TOXINS ON SOUTHWESTERN CORNBORER^(1,2) EG11063 EG11074 EG11091 EG11751 LC₅₀ 20 10-20 10-20 10-20EC₅₀ 0.2-2 0.2-2   0.2-2   0.2-2   ¹All values are expressed in μg/ml ofdiet. ²SWCB data ranges represent LC₅₀ and EC₅₀ ranges (as determined by% >1st instar), respectively.

TABLE 11 LC₅₀ VALUES OF CHIMERIC CRYSTAL PROTEINS ON SOYBEAN PESTS¹ PestEG11063 EG11074 EG11091 EG11751 EG11768 Velvetbean 0.9 0.6 0.3 0.1 0.06caterpillar¹ Soybean 0.9 0.8 0.6 0.7 0.2 looper ¹All values areexpressed in μg/ml of diet. ²Velvetbean caterpillar (Anticarsiagemmatalis) and soybean looper (Psuedoplusi includens) are both membersof the family Noctuidae.

6.6 EXAMPLE 6-AMINO ACID SEQUENCES OF THE NOVEL CRYSTAL PROTEINS 6.6.1AMINO ACID SEQUENCE OF THE EG11063 CRYSTAL PROTEIN (SEQ ID NO:10)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTryAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgllePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProvalGlyTyrvalThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.6.2 AMINO ACID SEQUENCE OF THE EG11074 CRYSTAL PROTEIN (SEQ IDNO:12)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAsnValThrAspTyrHisIleAspGlnValSerAsnLeuValThrTyrLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspSerAsnPheLysAspIleAsnArgGlnProGluArgGlyTrpGlyGlySerThrGlyIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgserValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.6.3 AMINO ACID SEQUENCE OF THE EG11735 CRYSTAL PROTEIN (SEQ IDNO:14)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrvalGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluGlySerIleArgSerProalsLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluvalTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysvalLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.6.4 AMINO ACID SEQUENCE OF THE EG11092 CRYSTAL PROTEIN (SEQ IDNO:26)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnproAlaLeuArgGluGluMetArgIleGlnpheAsnAspMetAsnserAjaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrArpHisAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrProIleArgThrValSerGluLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyvalTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrvalAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.6.5 AMINO ACID SEQUENCE OF THE EG11751 CRYSTAL PROTEIN (SEQ IDNO:28)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGLnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeugerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsnIleIleAlaSerAspSerIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGluTryIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.6.6 AMINO ACID SEQUENCE OF THE EG11091 CRYSTAL PROTEIN (SEQ IDNO:30)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaThrLeuThrAsnThrIleAspProGluArgIleAsnGlnIleProLeuValLysGlyPheArgValTrpGlyGlyThrSerValIleThrGlyProGlyPheThrGlyGlyAspIleLeuArgArgAsnThrPheGlyAspPheValSerLeuGlnValAsnIleAsnSerProIleThrGlnArgTyrArgLeuArgPheArgTyrAlaSerSerArgAspAlaArgValIleValLeuThrGlyAlaAlaSerThrGlyValGlyGlyGlnValSerValAsnMetProLeuGlnLysThrMetGluIleGlyGluAsnLeuThrSerArgThrPheArgTyrThrAspPheSerAsnProPheSerPheArgAlaAsnProAspIleIleGlyIleSerGluGlnProLeuPheGlyAlaGlySerIleSerSerGlyGluLeuTyrIleAspLysIleGluIleIleLeuAlaAspAlaThrPheGluAlaGluSerAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheArgGlyIleAsnArgGlnProAspArgGlyTrpArgGlySerThrAspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrValAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGluLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluIleValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysALaHisHisSerHisHisPheThrLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpvalIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuLeuGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGlyLysLeuGlnLeuGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhevalAsnSerGlnTyrAspArgLeuGlnValAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisArgIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaTyrSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuLeuCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeuValIleProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAspAsnThrAspGluLeuLysPheSerAsnCysValGLuGluGluVaLTyrProAsnAsnThrValThrCysAsnAsnTyrThrGlyThrGlnGluGluTyrGluGlyTHrTyrThrSerArgAsnGlnGluTyrAspGluAlaTyrGlyAsnAsnProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluSerAsnArgGlyTyrGlyAspTyrThrProLeuProAlaGlyTyrValThrLysAspLeuGluTyrPheProGLuThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.6.7 AMINO ACIDSEQUENCE OF THE EG11768 CRYSTAL PROTEIN (SEQ ID NO:34)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPhevalLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHIsLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrproIleArgThrValSerGluLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsnIleIleAlaSerAspSerIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyASnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAsnValThrAspTyrHisIleAspGlnValSerAsnLeuValThrTyrLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspSerAsnPheLysAspIleAsnArgGlnProGluArgGlyTrpGlyGlySerThrGlyIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.7 EXAMPLE 7-DNA SEQUENCES ENCODING THE NOVEL CRYSTAL PROTEINS6.7.1 DNA SEQUENCE ENCODING THE EG11063 CRYSTAL PROTEIN (SEQ ID NO:9)ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGTAAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACCCCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTTCCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTTGGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AACCAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGACTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCAGAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GACATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TATCAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCAGTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCGACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACAGAT TAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCGGAT TCT AGA 672 GAT TGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTAACT GTA 720 TTA GAT ATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TATCCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATCTAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATGGCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGAACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAGGGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATAGGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TATGGA ACC TCC TCA AAT TTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTAGAT TCG CTG GAT GAA ATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAAGGA TTT AGT CAT CGA TTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGTAAT AGT AGT GTA AGT ATA ATA 1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGTAGT GCA ACC CCT ACA AAT 1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCATTG GTA AAA GCA CAT 1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCCGGG TTT ACG GGA 1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCTTAT ACT ATT 1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGAATA CGC 1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGTGAA 1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTS GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGG ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA GGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 GCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531 6.7.2 DNA SEQUENCEENCODING THE EG11074 CRYSTAL PROTEIN (SEQ ID NO:11) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGO TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA GAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TGA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACAAAT 1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CCG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA CTC GAG GCT GAA TAT AAT CTG GAA AGA GCG GAG AAG GCG GTG1872 AAT GCG CTG TTT ACG TCT ACA AAC CAA CTA GGG CTA AAA ACA AAT GTA1920 ACG GAT TAT CAT ATT GAT CAA GTG TCC AAT TTA GTT ACG TAT TTA TCG1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAA CGC AAT TTA CTC CAA GAT TCA AAT2064 TTC AAA GAC ATT AAT AGG CAA CCA GAA CGT GGG TGG GGC GGA AGT ACA2112 GGG ATT ACC ATC CAA GGA GGG GAT GAC GTA TTT AAA GAA AAT TAC GTC2160 ACA CTA TCA GGT ACC TTT CAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA ATT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA GAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCO GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC ATG GAG GAA 3531 6.7.3 DNA SEQUENCE ENCODINGTHE EG11735 CRYSTAL PROTEIN (SEQ ID NO:13) ATG GAT AAC AAT CCG AAC ATCAAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAA GTA TTAGGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCC TTG TCGCTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GCT GCT GGA TTT GTG TTAGGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCATTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCTAGO AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATTTAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCA TTAAGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTT ACA ACCGCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTA TCA GTA480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 TATAAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT CAT GCT GTA 624 CGC TGGTAC AAT ACG GGA TTA GAG CGT GTA TGG GGA CCG CAT TCT AGA 672 GAT TGG ATAAGA TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GAT ATC GTTTCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA 768 ATT CGA ACA GTT TCCCAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTT GAT GGTAGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 GGA AGT ATT AGG AGT CCA CATTTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CAT AGA GGAGAA TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGG TTT TCGGGG CCA GGA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCACAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTA TCGTCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTATCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCATCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCGCCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGCCAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACA AAT1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG ACT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAP GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531 6.7.4 DNA SEQUENCEENCODING THE EG11092 CRYSTAL PROTEIN (SEQ ID NO:25) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GGT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT CAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAG CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG ATA AGA TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACAAAT 1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534 6.7.5 DNA SEQUENCEENCODING THE EG11751 CRYSTAL PROTEIN (SEQ ID NO:27) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA GTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT GAA TTT AATAAT 1392 ATA ATT GCA TCG GAT AGT ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT OGT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGP GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GPA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GPA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GPA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT CAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GPA TGG GPA GCA GPA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GPA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534 6.7.6 DNA SEQUENCEENCODING THE EG11091 CRYSTAL PROTEIN (SEQ ID NO:29) ATG GAT AAC AAT CCGAAC ATC ATT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GAA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGCAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAA CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT TTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCA ACT CTT ACAAAT 1392 ACA ATT GAT CCA GAG AGA ATT AAT CAA ATA CCT TTA GTG AAA GGA TTT1440 AGA GTT TGG GGG GGC ACC TCT GTC ATT ACA GGA CCA GGA TTT ACA GGA1488 GGG GAT ATC CTT CGA AGA AAT ACC TTT GGT GAT TTT GTA TCT CTA CAA1536 GTC AAT ATT AAT TCA CCA ATT ACC CAA AGA TAC CGT TTA AGA TTT CGT1584 TAC GCT TCC AGT AGG GAT GCA CGA GTT ATA GTA TTA ACA GGA GCG GCA1632 TCC ACA GGA GTC GGA GGC CAA GTT AGT GTA AAT ATG CCT CTT CAG AAA1680 ACT ATG GAA ATA GGG GAG AAC TTA ACA TCT AGA ACA TTT AGA TAT ACC1728 GAT TTT AGT AAT CCT TTT TCA TTT AGA GCT AAT CCA GAT ATA ATT GGG1776 ATA AGT GAA CAA CCT CTA TTT GGT GCA GGT TCT ATT AGT AGC GGT GAA1824 CTT TAT ATA GAT AAA ATT GAA ATT ATT CTA GCA GAT GCA ACA TTT GAA1872 GCA GAA TCT GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCC CTG TTT1920 ACT TCT TCC AAT CAA ATC GGG TTA AAA ACC GAT GTG ACG GAT TAT CAT1968 ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA TTT TGT2016 CTG GAT GAA AAG GGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA2064 CTC AGT GAT GAG CGG AAT TTA CGT CAA GAT CCA AAC TTC AGA GGG ATC2112 AAT AGA CAA CCA GAC CGT GGC TGG AGA GGA AGT ACA GAT ATT ACC ATC2160 CAA GGA GGA GAT GAC GTA TTC AAA GAG AAT TAC GTC ACA CTA CCG GGT2208 ACC GTT GAT GAG TGC TAT CCA ACG TAT TTA TAT CAG AAA ATA GAT GAG2256 TCG AAA TTA AAA GCT TAT ACC CGT TAT GAA TTA AGA GGG TAT ATC GAA2304 GAT AGT CAA GAC TTA GAA ATC TAT TTG ATC CGT TAC AAT GCA AAA CAC2352 GAA ATA GTA AAT GTG CCA GGC ACG GGT TCC TTA TGG CCG CTT TCA GCC2400 CAA AGT CCA ATC GGA AAG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC2448 CTT GAA TGG AAT CCT GAT CTA GAT TGT TCC TGC AGA GAC GGG GAA AAA2496 TGT GCA CAT CAT TCC CAT CAT TTC ACC TTG GAT ATT GAT GTT GGA TGT2544 ACA GAC TTA AAT GAG GAC TTA GGT GTA TGG GTG ATA TTC AAG ATT AAG2592 ACG CAA GAT GGC CAT GCA AGA CTA GGG AAT CTA GAG TTT CTC GAA GAG2640 AAA CCA TTA TTA GGG GAA GCA CTA GCT CGT GTG AAA AGA GCG GAG AAG2688 AAG TGG AGA GAC AAA CGA GAG AAA CTG CAG TTG GAA ACA AAT ATT GTT2736 TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA2784 TAT GAT AGA TTA CAA GTG GAT ACG AAC ATC GCA ATG ATT CAT GCG GCA2832 GAT AAA CGC GTT CAT AGA ATC CGG GAA GCG TAT CTG CCA GAG TTG TCT2880 GTG ATT CCA GGT GTC AAT GCG GCC ATT TTC GAA GAA TTA GAG GGA CGT2928 ATT TTT ACA GCG TAT TCC TTA TAT GAT GCG AGA AAT GTC ATT AAA AAT2976 GGC GAT TTC AAT AAT GGC TTA TTA TGC TGG AAC GTG AAA GGT CAT GTA3024 GAT GTA GAA GAG CAA AAC AAC CAC CGT TCG GTC CTT GTT ATC CCA GAA3072 TGG GAG GCA GAA GTA TCA CAA GAG GTT CGT GTC TGT CCA GGT CGT GGC3120 TAT ATC CTT CGT GTC ACA GCA TAT AAA GAG GGA TAT GGA GAG GGC TGC3168 GTA ACG ATC CAT GAG ATC GAA GCO AAT ACA GAC CAA CTG AAA TTC AGC3216 AAC TGT GTA GAA GAG GAA GTA TAT CCA AAC AAC ACA GTA ACG TGT AAT3264 AAT TAT ACT GGG ACT CCA GAA GAA TAT GAG GGT ACG TAC ACT TCT CGT3312 AAT CAA GGA TAT GAC GAA GCC TAT GGT AAT AAC CCT TCC GTA CCA GCT3360 GAT TAC GCT TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3408 GAG AAT CCT TGT GAA TCT AAC AGA GGC TAT GGG GAT TAC ACA CCA CTA3456 CCG GCT GGT TAT GTA ACA AAG GAT TTA GAG TAC TTC CCA GAG ACC GAT3504 AAG GTA TGG ATT GAG ATC GGA GAA ACA GAA GGA ACA TTC ATC GTG GAT3552 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3579 6.7.7 DNA SEQUENCEENCODING THE EG11768 CRYSTAL PROTEIN (SEQ ID NO:33) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCC GAT TCT AGA 672 GATTGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GAA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT GAA TTT AATAAT 1392 ATA ATT GCA TCG GAT AGT ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG ACT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA CTC GAG GCT GAA TAT AAT CTG GAA AGA GCG CAG AAG GCG GTG1872 AAT GCG CTG TTT ACG TCT ACA AAC CAA CTA GGG CTA AAA ACA AAT GTA1920 ACG GAT TAT CAT ATT GAT CAA GTG TCC AAT TTA GTT ACG TAT TTA TCG1968 GAT GAA TTT TGT GTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAA CGC AAT TTA CTC CAA GAT TCA AAT2064 TTC AAA GAC ATT AAT AGO CAA CCA GAA CGT GGG TOG GGC GGA AGT ACA2112 GGG ATT ACC ATC CAA GGA GGG GAT GAC GTA TTT AAA GAA AAT TAC GTC2160 ACA CTA TCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA CAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GOG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG ACA AAT2928 GTC ATT AAA AAT GGT CAT TTT AAT AAT GGG TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA CAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGG TAT ATC CTT CTT GTC ACA GCG TAC AAG GAG CGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTC GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 35 <210> SEQ ID NO 1 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 1ggatagcact catcaaaggt acc            #                  #                23 <210> SEQ ID NO 2 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 2gaagatatcc aattcgaaca gtttccc           #                  #             27 <210> SEQ ID NO 3 <211> LENGTH: 28 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 3catattctgc ctcgagtgtt gcagtaac          #                  #             28 <210> SEQ ID NO 4 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 4cccgatcggc cgcatgc              #                   #                  #   17 <210> SEQ ID NO 5 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 5cattggagct ctccatg              #                   #                  #   17 <210> SEQ ID NO 6 <211> LENGTH: 16 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 6gcactacgat gtatcc              #                   #                  #    16 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 7catcgtagtg caactcttac             #                  #                   # 20 <210> SEQ ID NO 8 <211> LENGTH: 39<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 8ccaagaaaat actagagctc ttgttaaaaa aggtgttcc       #                  #    39 <210> SEQ ID NO 9 <211> LENGTH: 3531 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <221> NAME/KEY: CDS<222> LOCATION: (1)..(3531) <400> SEQUENCE: 9atg gat aac aat ccg aac atc aat gaa tgc at#t cct tat aat tgt tta       48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15agt aac cct gaa gta gaa gta tta ggt gga ga#a aga ata gaa act ggt       96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30tac acc cca atc gat att tcc ttg tcg cta ac#g caa ttt ctt ttg agt      144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45gaa ttt gtt ccc ggt gct gga ttt gtg tta gg#a cta gtt gat ata ata      192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60tgg gga att ttt ggt ccc tct caa tgg gac gc#a ttt ctt gta caa att      240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80gaa cag tta att aac caa aga ata gaa gaa tt#c gct agg aac caa gcc      288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95att tct aga tta gaa gga cta agc aat ctt ta#t caa att tac gca gaa      336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110tct ttt aga gag tgg gaa gca gat cct act aa#t cca gca tta aga gaa      384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125gag atg cgt att caa ttc aat gac atg aac ag#t gcc ctt aca acc gct      432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140att cct ctt ttt gca gtt caa aat tat caa gt#t cct ctt tta tca gta      480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60tat gtt caa gct gca aat tta cat tta tca gt#t ttg aga gat gtt tca      528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175gtg ttt gga caa agg tgg gga ttt gat gcc gc#g act atc aat agt cgt      576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190tat aat gat tta act agg ctt att ggc aac ta#t aca gat tat gct gta      624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp Tyr Ala Val        195           #       200           #       205cgc tgg tac aat acg gga tta gaa cgt gta tg#g gga ccg gat tct aga      672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220gat tgg gta agg tat aat caa ttt aga aga ga#a tta aca cta act gta      720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40tta gat atc gtt gct ctg ttc ccg aat tat ga#t agt aga aga tat cca      768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As #p Ser Arg Arg Tyr Pro                245   #               250   #               255att cga aca gtt tcc caa tta aca aga gaa at#t tat aca aac cca gta      816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270tta gaa aat ttt gat ggt agt ttt cga ggc tc#g gct cag ggc ata gaa      864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285aga agt att agg agt cca cat ttg atg gat at#a ctt aac agt ata acc      912Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300atc tat acg gat gct cat agg ggt tat tat ta#t tgg tca ggg cat caa      960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20ata atg gct tct cct gta ggg ttt tcg ggg cc#a gaa ttc act ttt ccg     1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335cta tat gga act atg gga aat gca gct cca ca#a caa cgt att gtt gct     1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350caa cta ggt cag ggc gtg tat aga aca tta tc#g tcc act tta tat aga     1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365aga cct ttt aat ata ggg ata aat aat caa ca#a cta tct gtt ctt gac     1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380ggg aca gaa ttt gct tat gga acc tcc tca aa#t ttg cca tcc gct gta     1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00tac aga aaa agc gga acg gta gat tcg ctg ga#t gaa ata ccg cca cag     1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415aat aac aac gtg cca cct agg caa gga ttt ag#t cat cga tta agc cat     1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430gtt tca atg ttt cgt tca ggc ttt agt aat ag#t agt gta agt ata ata     1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445aga gct cca atg ttt tct tgg acg cac cgt ag#t gca acc cct aca aat     1392Arg Ala Pro Met Phe Ser Trp Thr His Arg Se #r Ala Thr Pro Thr Asn    450               #   455               #   460aca att gat ccg gag agg att act caa ata cc#a ttg gta aaa gca cat     1440Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80aca ctt cag tca ggt act act gtt gta aga gg#g ccc ggg ttt acg gga     1488Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495gga gat att ctt cga cga aca agt gga gga cc#a ttt gct tat act att     1536Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510gtt aat ata aat ggg caa tta ccc caa agg ta#t cgt gca aga ata cgc     1584Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525tat gcc tct act aca aat cta aga att tac gt#a acg gtt gca ggt gaa     1632Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540cgg att ttt gct ggt caa ttt aac aaa aca at#g gat acc ggt gac cca     1680Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60tta aca ttc caa tct ttt agt tac gca act at#t aat aca gct ttt aca     1728Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575ttc cca atg agc cag agt agt ttc aca gta gg#t gct gat act ttt agt     1776Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590tca ggg aat gaa gtt tat ata gac aga ttt ga#a ttg att cca gtt act     1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605gca aca ttt gaa gca gaa tat gat tta gaa ag#a gca caa aag gcg gtg     1872Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620aat gcg ctg ttt act tct ata aac caa ata gg#g ata aaa aca gat gtg     1920Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl #y Ile Lys Thr Asp Val625                 6 #30                 6 #35                 6 #40acg gat tat cat att gat caa gta tcc aat tt#a gtg gat tgt tta tca     1968Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Asp Cys Leu Ser                645   #               650   #               655gat gaa ttt tgt ctg gat gaa aag cga gaa tt#g tcc gag aaa gtc aaa     2016Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670cat gcg aag cga ctc agt gat gag cgg aat tt#a ctt caa gat cca aac     2064His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Pro Asn        675           #       680           #       685ttc aaa ggc atc aat agg caa cta gac cgt gg#t tgg aga gga agt acg     2112Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl #y Trp Arg Gly Ser Thr    690               #   695               #   700gat att acc atc caa aga gga gat gac gta tt#c aaa gaa aat tat gtc     2160Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20aca cta cca ggt acc ttt gat gag tgc tat cc#a aca tat ttg tat caa     2208Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735aaa atc gat gaa tca aaa tta aaa gcc ttt ac#c cgt tat caa tta aga     2256Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750ggg tat atc gaa gat agt caa gac tta gaa at#c tat tta att cgc tac     2304Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765aat gca aaa cat gaa aca gta aat gtg cca gg#t acg ggt tcc tta tgg     2352Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780ccg ctt tca gcc caa agt cca atc gga aag tg#t gga gag ccg aat cga     2400Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00tgc gcg cca cac ctt gaa tgg aat cct gac tt#a gat tgt tcg tgt agg     2448Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815gat gga gaa aag tgt gcc cat cat tcg cat ca#t ttc tcc tta gac att     2496Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830gat gta gga tgt aca gac tta aat gag gac ct#a ggt gta tgg gtg atc     2544Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845ttt aag att aag acg caa gat ggg cac gca ag#a cta ggg aat cta gag     2592Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860ttt ctc gaa gag aaa cca tta gta gga gaa gc#g cta gct cgt gtg aaa     2640Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80aga gcg gag aaa aaa tgg aga gac aaa cgt ga#a aaa ttg gaa tgg gaa     2688Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895aca aat atc gtt tat aaa gag gca aaa gaa tc#t gta gat gct tta ttt     2736Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910gta aac tct caa tat gat caa tta caa gcg ga#t acg aat att gcc atg     2784Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925att cat gcg gca gat aaa cgt gtt cat agc at#t cga gaa gct tat ctg     2832Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940cct gag ctg tct gtg att ccg ggt gtc aat gc#g gct att ttt gaa gaa     2880Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60tta gaa ggg cgt att ttc act gca ttc tcc ct#a tat gat gcg aga aat     2928Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975gtc att aaa aat ggt gat ttt aat aat ggc tt#a tcc tgc tgg aac gtg     2976Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990aaa ggg cat gta gat gta gaa gaa  caa aac #aac caa cgt  tcg gtc ctt   3024Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005gtt gtt  ccg gaa tgg gaa gca  gaa gtg t#ca caa gaa  gtt cgt gtc      3069Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020tgt ccg  ggt cgt ggc tat atc  ctt cgt g#tc aca gcg  tac aag gag      3114Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035gga tat  gga gaa ggt tgc gta  acc att c#at gag atc  gag aac aat      3159Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050aca gac  gaa ctg aag ttt agc  aac tgc g#ta gaa gag  gaa atc tat      3204Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065cca aat  aac acg gta acg tgt  aat gat t#at act gta  aat caa gaa      3249Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080gaa tac  gga ggt gcg tac act  tct cgt a#at cga gga  tat aac gaa      3294Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095gct cct  tcc gta cca gct gat  tat gcg t#ca gtc tat  gaa gaa aaa      3339Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110tcg tat  aca gat gga cga aga  gag aat c#ct tgt gaa  ttt aac aga      3384Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125ggg tat  agg gat tac acg cca  cta cca g#tt ggt tat  gtg aca aaa      3429Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140gaa tta  gaa tac ttc cca gaa  acc gat a#ag gta tgg  att gag att      3474Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155gga gaa  acg gaa gga aca ttt  atc gtg g#ac agc gtg  gaa tta ctc      3519Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170ctt atg  gag gaa            #                   #                  #     3531 Leu Met  Glu Glu     1175 <210> SEQ ID NO 10<211> LENGTH: 1177 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Hybrid Delta-Endotoxin<400> SEQUENCE: 10 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il#e Pro Tyr Asn Cys Leu 1               5    #                10  #                15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl#u Arg Ile Glu Thr Gly             20       #            25      #            30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th#r Gln Phe Leu Leu Ser         35           #        40          #        45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl#y Leu Val Asp Ile Ile     50               #    55              #    60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al#a Phe Leu Val Gln Ile 65                   #70                  #75                   #80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph#e Ala Arg Asn Gln Ala                 85   #                90  #                95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty#r Gln Ile Tyr Ala Glu             100       #           105      #           110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As#n Pro Ala Leu Arg Glu         115           #       120          #       125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se#r Ala Leu Thr Thr Ala     130               #   135              #   140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va#l Pro Leu Leu Ser Val 145                 1 #50                 1#55                 1 #60 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va#l Leu Arg Asp Val Ser                 165   #               170  #               175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al#a Thr Ile Asn Ser Arg             180       #           185      #           190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty#r Thr Asp Tyr Ala Val         195           #       200          #       205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr#p Gly Pro Asp Ser Arg     210               #   215              #   220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl#u Leu Thr Leu Thr Val 225                 2 #30                 2#35                 2 #40 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As#p Ser Arg Arg Tyr Pro                 245   #               250  #               255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il#e Tyr Thr Asn Pro Val             260       #           265      #           270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se#r Ala Gln Gly Ile Glu         275           #       280          #       285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Il#e Leu Asn Ser Ile Thr     290               #   295              #   300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty#r Trp Ser Gly His Gln 305                 3 #10                 3#15                 3 #20 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr#o Glu Phe Thr Phe Pro                 325   #               330  #               335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl#n Gln Arg Ile Val Ala             340       #           345      #           350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se#r Ser Thr Leu Tyr Arg         355           #       360          #       365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl#n Leu Ser Val Leu Asp     370               #   375              #   380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As#n Leu Pro Ser Ala Val 385                 3 #90                 3#95                 4 #00 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As#p Glu Ile Pro Pro Gln                 405   #               410  #               415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se#r His Arg Leu Ser His             420       #           425      #           430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se#r Ser Val Ser Ile Ile         435           #       440          #       445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Se#r Ala Thr Pro Thr Asn     450               #   455              #   460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr#o Leu Val Lys Ala His 465                 4 #70                 4#75                 4 #80 Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl#y Pro Gly Phe Thr Gly                 485   #               490  #               495 Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr#o Phe Ala Tyr Thr Ile             500       #           505      #           510 Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty#r Arg Ala Arg Ile Arg         515           #       520          #       525 Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va#l Thr Val Ala Gly Glu     530               #   535              #   540 Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me#t Asp Thr Gly Asp Pro 545                 5 #50                 5#55                 5 #60 Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il#e Asn Thr Ala Phe Thr                 565   #               570  #               575 Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl#y Ala Asp Thr Phe Ser             580       #           585      #           590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl#u Leu Ile Pro Val Thr         595           #       600          #       605 Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar#g Ala Gln Lys Ala Val     610               #   615              #   620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl#y Ile Lys Thr Asp Val 625                 6 #30                 6#35                 6 #40 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le#u Val Asp Cys Leu Ser                 645   #               650  #               655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le#u Ser Glu Lys Val Lys             660       #           665      #           670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le#u Leu Gln Asp Pro Asn         675           #       680          #       685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl#y Trp Arg Gly Ser Thr     690               #   695              #   700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph#e Lys Glu Asn Tyr Val 705                 7 #10                 7#15                 7 #20 Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr#o Thr Tyr Leu Tyr Gln                 725   #               730  #               735 Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th#r Arg Tyr Gln Leu Arg             740       #           745      #           750 Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il#e Tyr Leu Ile Arg Tyr         755           #       760          #       765 Asn Ala Lys His Glu Thr Val Asn Val Pro Gl#y Thr Gly Ser Leu Trp     770               #   775              #   780 Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy#s Gly Glu Pro Asn Arg 785                 7 #90                 7#95                 8 #00 Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le#u Asp Cys Ser Cys Arg                 805   #               810  #               815 Asp Gly Glu Lys Cys Ala His His Ser His Hi#s Phe Ser Leu Asp Ile             820       #           825      #           830 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le#u Gly Val Trp Val Ile         835           #       840          #       845 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar#g Leu Gly Asn Leu Glu     850               #   855              #   860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al#a Leu Ala Arg Val Lys 865                 8 #70                 8#75                 8 #80 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl#u Lys Leu Glu Trp Glu                 885   #               890  #               895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se#r Val Asp Ala Leu Phe             900       #           905      #           910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As#p Thr Asn Ile Ala Met         915           #       920          #       925 Ile His Ala Ala Asp Lys Arg Val His Ser Il#e Arg Glu Ala Tyr Leu     930               #   935              #   940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al#a Ala Ile Phe Glu Glu 945                 9 #50                 9#55                 9 #60 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le#u Tyr Asp Ala Arg Asn                 965   #               970  #               975 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le#u Ser Cys Trp Asn Val             980       #           985      #           990 Lys Gly His Val Asp Val Glu Glu  Gln Asn #Asn Gln Arg  Ser Val Leu         995           #       1000          #       1005 Val Val  Pro Glu Trp Glu Ala  Glu Val S#er Gln Glu  Val Arg Val     1010              #    1015             #    1020 Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V#al Thr Ala  Tyr Lys Glu     1025              #    1030             #    1035 Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H#is Glu Ile  Glu Asn Asn     1040              #    1045             #    1050 Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V#al Glu Glu  Glu Ile Tyr     1055              #    1060             #    1065 Pro Asn  Asn Thr Val Thr Cys  Asn Asp T#yr Thr Val  Asn Gln Glu     1070              #    1075             #    1080 Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A#sn Arg Gly  Tyr Asn Glu     1085              #    1090             #    1095 Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S#er Val Tyr  Glu Glu Lys     1100              #    1105             #    1110 Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P#ro Cys Glu  Phe Asn Arg     1115              #    1120             #    1125 Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V#al Gly Tyr  Val Thr Lys     1130              #    1135             #    1140 Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L#ys Val Trp  Ile Glu Ile     1145              #    1150             #    1155 Gly Glu  Thr Glu Gly Thr Phe  Ile Val A#sp Ser Val  Glu Leu Leu     1160              #    1165             #    1170 Leu Met  Glu Glu     1175 <210> SEQ ID NO 11<211> LENGTH: 3531 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Hybrid Delta-Endotoxin<221> NAME/KEY: CDS <222> LOCATION: (1)..(3531) <400> SEQUENCE: 11atg gat aac aat ccg aac atc aat gaa tgc at#t cct tat aat tgt tta       48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15agt aac cct gaa gta gaa gta tta ggt gga ga#a aga ata gaa act ggt       96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30tac acc cca atc gat att tcc ttg tcg cta ac#g caa ttt ctt ttg agt      144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45gaa ttt gtt ccc ggt gct gga ttt gtg tta gg#a cta gtt gat ata ata      192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60tgg gga att ttt ggt ccc tct caa tgg gac gc#a ttt ctt gta caa att      240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80gaa cag tta att aac caa aga ata gaa gaa tt#c gct agg aac caa gcc      288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95att tct aga tta gaa gga cta agc aat ctt ta#t caa att tac gca gaa      336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110tct ttt aga gag tgg gaa gca gat cct act aa#t cca gca tta aga gaa      384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125gag atg cgt att caa ttc aat gac atg aac ag#t gcc ctt aca acc gct      432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140att cct ctt ttt gca gtt caa aat tat caa gt#t cct ctt tta tca gta      480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60tat gtt caa gct gca aat tta cat tta tca gt#t ttg aga gat gtt tca      528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175gtg ttt gga caa agg tgg gga ttt gat gcc gc#g act atc aat agt cgt      576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190tat aat gat tta act agg ctt att ggc aac ta#t aca gat tat gct gta      624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp Tyr Ala Val        195           #       200           #       205cgc tgg tac aat acg gga tta gaa cgt gta tg#g gga ccg gat tct aga      672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220gat tgg gta agg tat aat caa ttt aga aga ga#a tta aca cta act gta      720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40tta gat atc gtt gct ctg ttc ccg aat tat ga#t agt aga aga tat cca      768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As #p Ser Arg Arg Tyr Pro                245   #               250   #               255att cga aca gtt tcc caa tta aca aga gaa at#t tat aca aac cca gta      816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270tta gaa aat ttt gat ggt agt ttt cga ggc tc#g gct cag ggc ata gaa      864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285aga agt att agg agt cca cat ttg atg gat at#a ctt aac agt ata acc      912Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300atc tat acg gat gct cat agg ggt tat tat ta#t tgg tca ggg cat caa      960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20ata atg gct tct cct gta ggg ttt tcg ggg cc#a gaa ttc act ttt ccg     1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335cta tat gga act atg gga aat gca gct cca ca#a caa cgt att gtt gct     1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350caa cta ggt cag ggc gtg tat aga aca tta tc#g tcc act tta tat aga     1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365aga cct ttt aat ata ggg ata aat aat caa ca#a cta tct gtt ctt gac     1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380ggg aca gaa ttt gct tat gga acc tcc tca aa#t ttg cca tcc gct gta     1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00tac aga aaa agc gga acg gta gat tcg ctg ga#t gaa ata ccg cca cag     1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415aat aac aac gtg cca cct agg caa gga ttt ag#t cat cga tta agc cat     1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430gtt tca atg ttt cgt tca ggc ttt agt aat ag#t agt gta agt ata ata     1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445aga gct cca atg ttt tct tgg acg cac cgt ag#t gca acc cct aca aat     1392Arg Ala Pro Met Phe Ser Trp Thr His Arg Se #r Ala Thr Pro Thr Asn    450               #   455               #   460aca att gat ccg gag agg att act caa ata cc#a ttg gta aaa gca cat     1440Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80aca ctt cag tca ggt act act gtt gta aga gg#g ccc ggg ttt acg gga     1488Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495gga gat att ctt cga cga aca agt gga gga cc#a ttt gct tat act att     1536Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510gtt aat ata aat ggg caa tta ccc caa agg ta#t cgt gca aga ata cgc     1584Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525tat gcc tct act aca aat cta aga att tac gt#a acg gtt gca ggt gaa     1632Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540cgg att ttt gct ggt caa ttt aac aaa aca at#g gat acc ggt gac cca     1680Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60tta aca ttc caa tct ttt agt tac gca act at#t aat aca gct ttt aca     1728Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575ttc cca atg agc cag agt agt ttc aca gta gg#t gct gat act ttt agt     1776Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590tca ggg aat gaa gtt tat ata gac aga ttt ga#a ttg att cca gtt act     1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605gca aca ttt gaa gca gaa tat gat tta gaa ag#a gca caa aag gcg gtg     1872Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620aat gcg ctg ttt act tct ata aac caa ata gg#g ata aaa aca gat gtg     1920Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl #y Ile Lys Thr Asp Val625                 6 #30                 6 #35                 6 #40acg gat tat cat att gat caa gta tcc aat tt#a gtg gat tgt tta tca     1968Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Asp Cys Leu Ser                645   #               650   #               655gat gaa ttt tgt ctg gat gaa aag cga gaa tt#g tcc gag aaa gtc aaa     2016Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670cat gcg aag cga ctc agt gat gag cgg aat tt#a ctt caa gat cca aac     2064His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Pro Asn        675           #       680           #       685ttc aaa ggc atc aat agg caa cta gac cgt gg#t tgg aga gga agt acg     2112Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl #y Trp Arg Gly Ser Thr    690               #   695               #   700gat att acc atc caa aga gga gat gac gta tt#c aaa gaa aat tat gtc     2160Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20aca cta cca ggt acc ttt gat gag tgc tat cc#a aca tat ttg tat caa     2208Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735aaa atc gat gaa tca aaa tta aaa gcc ttt ac#c cgt tat caa tta aga     2256Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750ggg tat atc gaa gat agt caa gac tta gaa at#c tat tta att cgc tac     2304Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765aat gca aaa cat gaa aca gta aat gtg cca gg#t acg ggt tcc tta tgg     2352Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780ccg ctt tca gcc caa agt cca atc gga aag tg#t gga gag ccg aat cga     2400Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00tgc gcg cca cac ctt gaa tgg aat cct gac tt#a gat tgt tcg tgt agg     2448Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815gat gga gaa aag tgt gcc cat cat tcg cat ca#t ttc tcc tta gac att     2496Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830gat gta gga tgt aca gac tta aat gag gac ct#a ggt gta tgg gtg atc     2544Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845ttt aag att aag acg caa gat ggg cac gca ag#a cta ggg aat cta gag     2592Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860ttt ctc gaa gag aaa cca tta gta gga gaa gc#g cta gct cgt gtg aaa     2640Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80aga gcg gag aaa aaa tgg aga gac aaa cgt ga#a aaa ttg gaa tgg gaa     2688Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895aca aat atc gtt tat aaa gag gca aaa gaa tc#t gta gat gct tta ttt     2736Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910gta aac tct caa tat gat caa tta caa gcg ga#t acg aat att gcc atg     2784Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925att cat gcg gca gat aaa cgt gtt cat agc at#t cga gaa gct tat ctg     2832Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940cct gag ctg tct gtg att ccg ggt gtc aat gc#g gct att ttt gaa gaa     2880Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60tta gaa ggg cgt att ttc act gca ttc tcc ct#a tat gat gcg aga aat     2928Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975gtc att aaa aat ggt gat ttt aat aat ggc tt#a tcc tgc tgg aac gtg     2976Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990aaa ggg cat gta gat gta gaa gaa  caa aac #aac caa cgt  tcg gtc ctt   3024Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005gtt gtt  ccg gaa tgg gaa gca  gaa gtg t#ca caa gaa  gtt cgt gtc      3069Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020tgt ccg  ggt cgt ggc tat atc  ctt cgt g#tc aca gcg  tac aag gag      3114Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035gga tat  gga gaa ggt tgc gta  acc att c#at gag atc  gag aac aat      3159Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050aca gac  gaa ctg aag ttt agc  aac tgc g#ta gaa gag  gaa atc tat      3204Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065cca aat  aac acg gta acg tgt  aat gat t#at act gta  aat caa gaa      3249Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080gaa tac  gga ggt gcg tac act  tct cgt a#at cga gga  tat aac gaa      3294Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095gct cct  tcc gta cca gct gat  tat gcg t#ca gtc tat  gaa gaa aaa      3339Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110tcg tat  aca gat gga cga aga  gag aat c#ct tgt gaa  ttt aac aga      3384Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125ggg tat  agg gat tac acg cca  cta cca g#tt ggt tat  gtg aca aaa      3429Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140gaa tta  gaa tac ttc cca gaa  acc gat a#ag gta tgg  att gag att      3474Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155gga gaa  acg gaa gga aca ttt  atc gtg g#ac agc gtg  gaa tta ctc      3519Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170ctt atg  gag gaa            #                   #                  #     3531 Leu Met  Glu Glu     1175 <210> SEQ ID NO 12<211> LENGTH: 1177 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Hybrid Delta-Endotoxin<400> SEQUENCE: 12 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il#e Pro Tyr Asn Cys Leu 1               5    #                10  #                15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl#u Arg Ile Glu Thr Gly             20       #            25      #            30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th#r Gln Phe Leu Leu Ser         35           #        40          #        45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl#y Leu Val Asp Ile Ile     50               #    55              #    60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al#a Phe Leu Val Gln Ile 65                   #70                  #75                   #80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph#e Ala Arg Asn Gln Ala                 85   #                90  #                95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty#r Gln Ile Tyr Ala Glu             100       #           105      #           110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As#n Pro Ala Leu Arg Glu         115           #       120          #       125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se#r Ala Leu Thr Thr Ala     130               #   135              #   140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va#l Pro Leu Leu Ser Val 145                 1 #50                 1#55                 1 #60 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va#l Leu Arg Asp Val Ser                 165   #               170  #               175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al#a Thr Ile Asn Ser Arg             180       #           185      #           190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty#r Thr Asp Tyr Ala Val         195           #       200          #       205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr#p Gly Pro Asp Ser Arg     210               #   215              #   220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl#u Leu Thr Leu Thr Val 225                 2 #30                 2#35                 2 #40 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As#p Ser Arg Arg Tyr Pro                 245   #               250  #               255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il#e Tyr Thr Asn Pro Val             260       #           265      #           270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se#r Ala Gln Gly Ile Glu         275           #       280          #       285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Il#e Leu Asn Ser Ile Thr     290               #   295              #   300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty#r Trp Ser Gly His Gln 305                 3 #10                 3#15                 3 #20 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr#o Glu Phe Thr Phe Pro                 325   #               330  #               335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl#n Gln Arg Ile Val Ala             340       #           345      #           350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se#r Ser Thr Leu Tyr Arg         355           #       360          #       365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl#n Leu Ser Val Leu Asp     370               #   375              #   380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As#n Leu Pro Ser Ala Val 385                 3 #90                 3#95                 4 #00 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As#p Glu Ile Pro Pro Gln                 405   #               410  #               415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se#r His Arg Leu Ser His             420       #           425      #           430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se#r Ser Val Ser Ile Ile         435           #       440          #       445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Se#r Ala Thr Pro Thr Asn     450               #   455              #   460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr#o Leu Val Lys Ala His 465                 4 #70                 4#75                 4 #80 Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl#y Pro Gly Phe Thr Gly                 485   #               490  #               495 Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr#o Phe Ala Tyr Thr Ile             500       #           505      #           510 Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty#r Arg Ala Arg Ile Arg         515           #       520          #       525 Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va#l Thr Val Ala Gly Glu     530               #   535              #   540 Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me#t Asp Thr Gly Asp Pro 545                 5 #50                 5#55                 5 #60 Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il#e Asn Thr Ala Phe Thr                 565   #               570  #               575 Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl#y Ala Asp Thr Phe Ser             580       #           585      #           590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl#u Leu Ile Pro Val Thr         595           #       600          #       605 Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar#g Ala Gln Lys Ala Val     610               #   615              #   620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl#y Ile Lys Thr Asp Val 625                 6 #30                 6#35                 6 #40 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le#u Val Asp Cys Leu Ser                 645   #               650  #               655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le#u Ser Glu Lys Val Lys             660       #           665      #           670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le#u Leu Gln Asp Pro Asn         675           #       680          #       685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl#y Trp Arg Gly Ser Thr     690               #   695              #   700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph#e Lys Glu Asn Tyr Val 705                 7 #10                 7#15                 7 #20 Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr#o Thr Tyr Leu Tyr Gln                 725   #               730  #               735 Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th#r Arg Tyr Gln Leu Arg             740       #           745      #           750 Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il#e Tyr Leu Ile Arg Tyr         755           #       760          #       765 Asn Ala Lys His Glu Thr Val Asn Val Pro Gl#y Thr Gly Ser Leu Trp     770               #   775              #   780 Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy#s Gly Glu Pro Asn Arg 785                 7 #90                 7#95                 8 #00 Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le#u Asp Cys Ser Cys Arg                 805   #               810  #               815 Asp Gly Glu Lys Cys Ala His His Ser His Hi#s Phe Ser Leu Asp Ile             820       #           825      #           830 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le#u Gly Val Trp Val Ile         835           #       840          #       845 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar#g Leu Gly Asn Leu Glu     850               #   855              #   860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al#a Leu Ala Arg Val Lys 865                 8 #70                 8#75                 8 #80 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl#u Lys Leu Glu Trp Glu                 885   #               890  #               895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se#r Val Asp Ala Leu Phe             900       #           905      #           910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As#p Thr Asn Ile Ala Met         915           #       920          #       925 Ile His Ala Ala Asp Lys Arg Val His Ser Il#e Arg Glu Ala Tyr Leu     930               #   935              #   940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al#a Ala Ile Phe Glu Glu 945                 9 #50                 9#55                 9 #60 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le#u Tyr Asp Ala Arg Asn                 965   #               970  #               975 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le#u Ser Cys Trp Asn Val             980       #           985      #           990 Lys Gly His Val Asp Val Glu Glu  Gln Asn #Asn Gln Arg  Ser Val Leu         995           #       1000          #       1005 Val Val  Pro Glu Trp Glu Ala  Glu Val S#er Gln Glu  Val Arg Val     1010              #    1015             #    1020 Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V#al Thr Ala  Tyr Lys Glu     1025              #    1030             #    1035 Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H#is Glu Ile  Glu Asn Asn     1040              #    1045             #    1050 Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V#al Glu Glu  Glu Ile Tyr     1055              #    1060             #    1065 Pro Asn  Asn Thr Val Thr Cys  Asn Asp T#yr Thr Val  Asn Gln Glu     1070              #    1075             #    1080 Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A#sn Arg Gly  Tyr Asn Glu     1085              #    1090             #    1095 Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S#er Val Tyr  Glu Glu Lys     1100              #    1105             #    1110 Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P#ro Cys Glu  Phe Asn Arg     1115              #    1120             #    1125 Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V#al Gly Tyr  Val Thr Lys     1130              #    1135             #    1140 Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L#ys Val Trp  Ile Glu Ile     1145              #    1150             #    1155 Gly Glu  Thr Glu Gly Thr Phe  Ile Val A#sp Ser Val  Glu Leu Leu     1160              #    1165             #    1170 Leu Met  Glu Glu     1175 <210> SEQ ID NO 13<211> LENGTH: 3531 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Hybrid Delta-Endotoxin<221> NAME/KEY: CDS <222> LOCATION: (1)..(3531) <400> SEQUENCE: 13atg gat aac aat ccg aac atc aat gaa tgc at#t cct tat aat tgt tta       48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15agt aac cct gaa gta gaa gta tta ggt gga ga#a aga ata gaa act ggt       96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30tac acc cca atc gat att tcc ttg tcg cta ac#g caa ttt ctt ttg agt      144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45gaa ttt gtt ccc ggt gct gga ttt gtg tta gg#a cta gtt gat ata ata      192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60tgg gga att ttt ggt ccc tct caa tgg gac gc#a ttt ctt gta caa att      240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80gaa cag tta att aac caa aga ata gaa gaa tt#c gct agg aac caa gcc      288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95att tct aga tta gaa gga cta agc aat ctt ta#t caa att tac gca gaa      336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110tct ttt aga gag tgg gaa gca gat cct act aa#t cca gca tta aga gaa      384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125gag atg cgt att caa ttc aat gac atg aac ag#t gcc ctt aca acc gct      432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140att cct ctt ttt gca gtt caa aat tat caa gt#t cct ctt tta tca gta      480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60tat gtt caa gct gca aat tta cat tta tca gt#t ttg aga gat gtt tca      528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175gtg ttt gga caa agg tgg gga ttt gat gcc gc#g act atc aat agt cgt      576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190tat aat gat tta act agg ctt att ggc aac ta#t aca gat tat gct gta      624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp Tyr Ala Val        195           #       200           #       205cgc tgg tac aat acg gga tta gaa cgt gta tg#g gga ccg gat tct aga      672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220gat tgg gta agg tat aat caa ttt aga aga ga#a tta aca cta act gta      720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40tta gat atc gtt gct ctg ttc ccg aat tat ga#t agt aga aga tat cca      768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As #p Ser Arg Arg Tyr Pro                245   #               250   #               255att cga aca gtt tcc caa tta aca aga gaa at#t tat aca aac cca gta      816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270tta gaa aat ttt gat ggt agt ttt cga ggc tc#g gct cag ggc ata gaa      864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285aga agt att agg agt cca cat ttg atg gat at#a ctt aac agt ata acc      912Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300atc tat acg gat gct cat agg ggt tat tat ta#t tgg tca ggg cat caa      960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20ata atg gct tct cct gta ggg ttt tcg ggg cc#a gaa ttc act ttt ccg     1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335cta tat gga act atg gga aat gca gct cca ca#a caa cgt att gtt gct     1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350caa cta ggt cag ggc gtg tat aga aca tta tc#g tcc act tta tat aga     1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365aga cct ttt aat ata ggg ata aat aat caa ca#a cta tct gtt ctt gac     1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380ggg aca gaa ttt gct tat gga acc tcc tca aa#t ttg cca tcc gct gta     1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00tac aga aaa agc gga acg gta gat tcg ctg ga#t gaa ata ccg cca cag     1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415aat aac aac gtg cca cct agg caa gga ttt ag#t cat cga tta agc cat     1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430gtt tca atg ttt cgt tca ggc ttt agt aat ag#t agt gta agt ata ata     1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445aga gct cca atg ttt tct tgg acg cac cgt ag#t gca acc cct aca aat     1392Arg Ala Pro Met Phe Ser Trp Thr His Arg Se #r Ala Thr Pro Thr Asn    450               #   455               #   460aca att gat ccg gag agg att act caa ata cc#a ttg gta aaa gca cat     1440Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80aca ctt cag tca ggt act act gtt gta aga gg#g ccc ggg ttt acg gga     1488Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495gga gat att ctt cga cga aca agt gga gga cc#a ttt gct tat act att     1536Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510gtt aat ata aat ggg caa tta ccc caa agg ta#t cgt gca aga ata cgc     1584Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525tat gcc tct act aca aat cta aga att tac gt#a acg gtt gca ggt gaa     1632Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540cgg att ttt gct ggt caa ttt aac aaa aca at#g gat acc ggt gac cca     1680Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60tta aca ttc caa tct ttt agt tac gca act at#t aat aca gct ttt aca     1728Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575ttc cca atg agc cag agt agt ttc aca gta gg#t gct gat act ttt agt     1776Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590tca ggg aat gaa gtt tat ata gac aga ttt ga#a ttg att cca gtt act     1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605gca aca ttt gaa gca gaa tat gat tta gaa ag#a gca caa aag gcg gtg     1872Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620aat gcg ctg ttt act tct ata aac caa ata gg#g ata aaa aca gat gtg     1920Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl #y Ile Lys Thr Asp Val625                 6 #30                 6 #35                 6 #40acg gat tat cat att gat caa gta tcc aat tt#a gtg gat tgt tta tca     1968Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Asp Cys Leu Ser                645   #               650   #               655gat gaa ttt tgt ctg gat gaa aag cga gaa tt#g tcc gag aaa gtc aaa     2016Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670cat gcg aag cga ctc agt gat gag cgg aat tt#a ctt caa gat cca aac     2064His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Pro Asn        675           #       680           #       685ttc aaa ggc atc aat agg caa cta gac cgt gg#t tgg aga gga agt acg     2112Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl #y Trp Arg Gly Ser Thr    690               #   695               #   700gat att acc atc caa aga gga gat gac gta tt#c aaa gaa aat tat gtc     2160Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20aca cta cca ggt acc ttt gat gag tgc tat cc#a aca tat ttg tat caa     2208Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735aaa atc gat gaa tca aaa tta aaa gcc ttt ac#c cgt tat caa tta aga     2256Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750ggg tat atc gaa gat agt caa gac tta gaa at#c tat tta att cgc tac     2304Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765aat gca aaa cat gaa aca gta aat gtg cca gg#t acg ggt tcc tta tgg     2352Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780ccg ctt tca gcc caa agt cca atc gga aag tg#t gga gag ccg aat cga     2400Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00tgc gcg cca cac ctt gaa tgg aat cct gac tt#a gat tgt tcg tgt agg     2448Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815gat gga gaa aag tgt gcc cat cat tcg cat ca#t ttc tcc tta gac att     2496Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830gat gta gga tgt aca gac tta aat gag gac ct#a ggt gta tgg gtg atc     2544Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845ttt aag att aag acg caa gat ggg cac gca ag#a cta ggg aat cta gag     2592Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860ttt ctc gaa gag aaa cca tta gta gga gaa gc#g cta gct cgt gtg aaa     2640Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80aga gcg gag aaa aaa tgg aga gac aaa cgt ga#a aaa ttg gaa tgg gaa     2688Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895aca aat atc gtt tat aaa gag gca aaa gaa tc#t gta gat gct tta ttt     2736Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910gta aac tct caa tat gat caa tta caa gcg ga#t acg aat att gcc atg     2784Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925att cat gcg gca gat aaa cgt gtt cat agc at#t cga gaa gct tat ctg     2832Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940cct gag ctg tct gtg att ccg ggt gtc aat gc#g gct att ttt gaa gaa     2880Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60tta gaa ggg cgt att ttc act gca ttc tcc ct#a tat gat gcg aga aat     2928Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975gtc att aaa aat ggt gat ttt aat aat ggc tt#a tcc tgc tgg aac gtg     2976Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990aaa ggg cat gta gat gta gaa gaa  caa aac #aac caa cgt  tcg gtc ctt   3024Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005gtt gtt  ccg gaa tgg gaa gca  gaa gtg t#ca caa gaa  gtt cgt gtc      3069Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020tgt ccg  ggt cgt ggc tat atc  ctt cgt g#tc aca gcg  tac aag gag      3114Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035gga tat  gga gaa ggt tgc gta  acc att c#at gag atc  gag aac aat      3159Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050aca gac  gaa ctg aag ttt agc  aac tgc g#ta gaa gag  gaa atc tat      3204Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065cca aat  aac acg gta acg tgt  aat gat t#at act gta  aat caa gaa      3249Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080gaa tac  gga ggt gcg tac act  tct cgt a#at cga gga  tat aac gaa      3294Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095gct cct  tcc gta cca gct gat  tat gcg t#ca gtc tat  gaa gaa aaa      3339Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110tcg tat  aca gat gga cga aga  gag aat c#ct tgt gaa  ttt aac aga      3384Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125ggg tat  agg gat tac acg cca  cta cca g#tt ggt tat  gtg aca aaa      3429Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140gaa tta  gaa tac ttc cca gaa  acc gat a#ag gta tgg  att gag att      3474Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155gga gaa  acg gaa gga aca ttt  atc gtg g#ac agc gtg  gaa tta ctc      3519Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170ctt atg  gag gaa            #                   #                  #     3531 Leu Met  Glu Glu     1175 <210> SEQ ID NO 14<211> LENGTH: 1177 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Hybrid Delta-Endotoxin<400> SEQUENCE: 14 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il#e Pro Tyr Asn Cys Leu 1               5    #                10  #                15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl#u Arg Ile Glu Thr Gly             20       #            25      #            30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th#r Gln Phe Leu Leu Ser         35           #        40          #        45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl#y Leu Val Asp Ile Ile     50               #    55              #    60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al#a Phe Leu Val Gln Ile 65                   #70                  #75                   #80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph#e Ala Arg Asn Gln Ala                 85   #                90  #                95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty#r Gln Ile Tyr Ala Glu             100       #           105      #           110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As#n Pro Ala Leu Arg Glu         115           #       120          #       125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se#r Ala Leu Thr Thr Ala     130               #   135              #   140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va#l Pro Leu Leu Ser Val 145                 1 #50                 1#55                 1 #60 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va#l Leu Arg Asp Val Ser                 165   #               170  #               175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al#a Thr Ile Asn Ser Arg             180       #           185      #           190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty#r Thr Asp Tyr Ala Val         195           #       200          #       205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr#p Gly Pro Asp Ser Arg     210               #   215              #   220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl#u Leu Thr Leu Thr Val 225                 2 #30                 2#35                 2 #40 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As#p Ser Arg Arg Tyr Pro                 245   #               250  #               255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il#e Tyr Thr Asn Pro Val             260       #           265      #           270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se#r Ala Gln Gly Ile Glu         275           #       280          #       285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Il#e Leu Asn Ser Ile Thr     290               #   295              #   300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty#r Trp Ser Gly His Gln 305                 3 #10                 3#15                 3 #20 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr#o Glu Phe Thr Phe Pro                 325   #               330  #               335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl#n Gln Arg Ile Val Ala             340       #           345      #           350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se#r Ser Thr Leu Tyr Arg         355           #       360          #       365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl#n Leu Ser Val Leu Asp     370               #   375              #   380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As#n Leu Pro Ser Ala Val 385                 3 #90                 3#95                 4 #00 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As#p Glu Ile Pro Pro Gln                 405   #               410  #               415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se#r His Arg Leu Ser His             420       #           425      #           430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se#r Ser Val Ser Ile Ile         435           #       440          #       445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Se#r Ala Thr Pro Thr Asn     450               #   455              #   460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr#o Leu Val Lys Ala His 465                 4 #70                 4#75                 4 #80 Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl#y Pro Gly Phe Thr Gly                 485   #               490  #               495 Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr#o Phe Ala Tyr Thr Ile             500       #           505      #           510 Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty#r Arg Ala Arg Ile Arg         515           #       520          #       525 Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va#l Thr Val Ala Gly Glu     530               #   535              #   540 Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me#t Asp Thr Gly Asp Pro 545                 5 #50                 5#55                 5 #60 Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il#e Asn Thr Ala Phe Thr                 565   #               570  #               575 Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl#y Ala Asp Thr Phe Ser             580       #           585      #           590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl#u Leu Ile Pro Val Thr         595           #       600          #       605 Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar#g Ala Gln Lys Ala Val     610               #   615              #   620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl#y Ile Lys Thr Asp Val 625                 6 #30                 6#35                 6 #40 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le#u Val Asp Cys Leu Ser                 645   #               650  #               655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le#u Ser Glu Lys Val Lys             660       #           665      #           670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le#u Leu Gln Asp Pro Asn         675           #       680          #       685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl#y Trp Arg Gly Ser Thr     690               #   695              #   700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph#e Lys Glu Asn Tyr Val 705                 7 #10                 7#15                 7 #20 Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr#o Thr Tyr Leu Tyr Gln                 725   #               730  #               735 Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th#r Arg Tyr Gln Leu Arg             740       #           745      #           750 Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il#e Tyr Leu Ile Arg Tyr         755           #       760          #       765 Asn Ala Lys His Glu Thr Val Asn Val Pro Gl#y Thr Gly Ser Leu Trp     770               #   775              #   780 Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy#s Gly Glu Pro Asn Arg 785                 7 #90                 7#95                 8 #00 Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le#u Asp Cys Ser Cys Arg                 805   #               810  #               815 Asp Gly Glu Lys Cys Ala His His Ser His Hi#s Phe Ser Leu Asp Ile             820       #           825      #           830 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le#u Gly Val Trp Val Ile         835           #       840          #       845 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar#g Leu Gly Asn Leu Glu     850               #   855              #   860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al#a Leu Ala Arg Val Lys 865                 8 #70                 8#75                 8 #80 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl#u Lys Leu Glu Trp Glu                 885   #               890  #               895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se#r Val Asp Ala Leu Phe             900       #           905      #           910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As#p Thr Asn Ile Ala Met         915           #       920          #       925 Ile His Ala Ala Asp Lys Arg Val His Ser Il#e Arg Glu Ala Tyr Leu     930               #   935              #   940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al#a Ala Ile Phe Glu Glu 945                 9 #50                 9#55                 9 #60 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le#u Tyr Asp Ala Arg Asn                 965   #               970  #               975 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le#u Ser Cys Trp Asn Val             980       #           985      #           990 Lys Gly His Val Asp Val Glu Glu  Gln Asn #Asn Gln Arg  Ser Val Leu         995           #       1000          #       1005 Val Val  Pro Glu Trp Glu Ala  Glu Val S#er Gln Glu  Val Arg Val     1010              #    1015             #    1020 Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V#al Thr Ala  Tyr Lys Glu     1025              #    1030             #    1035 Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H#is Glu Ile  Glu Asn Asn     1040              #    1045             #    1050 Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V#al Glu Glu  Glu Ile Tyr     1055              #    1060             #    1065 Pro Asn  Asn Thr Val Thr Cys  Asn Asp T#yr Thr Val  Asn Gln Glu     1070              #    1075             #    1080 Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A#sn Arg Gly  Tyr Asn Glu     1085              #    1090             #    1095 Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S#er Val Tyr  Glu Glu Lys     1100              #    1105             #    1110 Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P#ro Cys Glu  Phe Asn Arg     1115              #    1120             #    1125 Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V#al Gly Tyr  Val Thr Lys     1130              #    1135             #    1140 Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L#ys Val Trp  Ile Glu Ile     1145              #    1150             #    1155 Gly Glu  Thr Glu Gly Thr Phe  Ile Val A#sp Ser Val  Glu Leu Leu     1160              #    1165             #    1170 Leu Met  Glu Glu     1175 <210> SEQ ID NO 15 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 15tatccaattc gaacgtcatc             #                  #                   # 20 <210> SEQ ID NO 16 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 16tttagtcatc gattaaatca             #                  #                   # 20 <210> SEQ ID NO 17 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 17ataataagag ctccaatgtt             #                  #                   # 20 <210> SEQ ID NO 18 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 18tacatcgtag tgcaactctt             #                  #                   # 20 <210> SEQ ID NO 19 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 19tcatggagag ctcctatgtt             #                  #                   # 20 <210> SEQ ID NO 20 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 20ttaacaagag ctcctatgtt             #                  #                   # 20 <210> SEQ ID NO 21 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 21actaccaggt acctttgatg             #                  #                   # 20 <210> SEQ ID NO 22 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 22actaccgggt acctttgata             #                  #                   # 20 <210> SEQ ID NO 23 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 23atttgagtaa tactatcc              #                   #                  #  18 <210> SEQ ID NO 24 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 24attactcaaa taccattgg              #                  #                   # 19 <210> SEQ ID NO 25 <211> LENGTH: 3534<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <221> NAME/KEY: CDS<222> LOCATION: (1)..(3531) <400> SEQUENCE: 25atg gat aac aat ccg aac atc aat gaa tgc at#t cct tat aat tgt tta       48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15agt aac cct gaa gta gaa gta tta ggt gga ga#a aga ata gaa act ggt       96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30tac acc cca atc gat att tcc ttg tcg cta ac#g caa ttt ctt ttg agt      144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45gaa ttt gtt ccc ggt gct gga ttt gtg tta gg#a cta gtt gat ata ata      192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60tgg gga att ttt ggt ccc tct caa tgg gac gc#a ttt ctt gta caa att      240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80gaa cag tta att aac caa aga ata gaa gaa tt#c gct agg aac caa gcc      288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95att tct aga tta gaa gga cta agc aat ctt ta#t caa att tac gca gaa      336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110tct ttt aga gag tgg gaa gca gat cct act aa#t cca gca tta aga gaa      384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125gag atg cgt att caa ttc aat gac atg aac ag#t gcc ctt aca acc gct      432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140att cct ctt ttt gca gtt caa aat tat caa gt#t cct ctt tta tca gta      480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60tat gtt caa gct gca aat tta cat tta tca gt#t ttg aga gat gtt tca      528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175gtg ttt gga caa agg tgg gga ttt gat gcc gc#g act atc aat agt cgt      576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190tat aat gat tta act agg ctt att ggc aac ta#t aca gat cat gct gta      624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp His Ala Val        195           #       200           #       205cgc tgg tac aat acg gga tta gag cgt gta tg#g gga ccg gat tct aga      672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220gat tgg ata aga tat aat caa ttt aga aga ga#a tta aca cta act gta      720Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40tta gat atc gtt tct cta ttt ccg aac tat ga#t agt aga acg tat cca      768Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As #p Ser Arg Thr Tyr Pro                245   #               250   #               255att cga aca gtt tcc caa tta aca aga gaa at#t tat aca aac cca gta      816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270tta gaa aat ttt gat ggt agt ttt cga ggc tc#g gct cag ggc ata gaa      864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285aga agt att agg agt cca cat ttg atg gat at#a ctt aac agt ata acc      912Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300atc tat acg gat gct cat agg ggt tat tat ta#t tgg tca ggg cat caa      960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20ata atg gct tct cct gta ggg ttt tcg ggg cc#a gaa ttc act ttt ccg     1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335cta tat gga act atg gga aat gca gct cca ca#a caa cgt att gtt gct     1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350caa cta ggt cag ggc gtg tat aga aca tta tc#g tcc act tta tat aga     1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365aga cct ttt aat ata ggg ata aat aat caa ca#a cta tct gtt ctt gac     1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380ggg aca gaa ttt gct tat gga acc tcc tca aa#t ttg cca tcc gct gta     1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00tac aga aaa agc gga acg gta gat tcg ctg ga#t gaa ata ccg cca cag     1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415aat aac aac gtg cca cct agg caa gga ttt ag#t cat cga tta agc cat     1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430gtt tca atg ttt cgt tca ggc ttt agt aat ag#t agt gta agt ata ata     1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445aga gct cca atg ttt tct tgg acg cac cgt ag#t gca acc cct aca aat     1392Arg Ala Pro Met Phe Ser Trp Thr His Arg Se #r Ala Thr Pro Thr Asn    450               #   455               #   460aca att gat ccg gag agg att act caa ata cc#a ttg gta aaa gca cat     1440Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80aca ctt cag tca ggt act act gtt gta aga gg#g ccc ggg ttt acg gga     1488Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495gga gat att ctt cga cga aca agt gga gga cc#a ttt gct tat act att     1536Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510gtt aat ata aat ggg caa tta ccc caa agg ta#t cgt gca aga ata cgc     1584Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525tat gcc tct act aca aat cta aga att tac gt#a acg gtt gca ggt gaa     1632Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540cgg att ttt gct ggt caa ttt aac aaa aca at#g gat acc ggt gac cca     1680Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60tta aca ttc caa tct ttt agt tac gca act at#t aat aca gct ttt aca     1728Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575ttc cca atg agc cag agt agt ttc aca gta gg#t gct gat act ttt agt     1776Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590tca ggg aat gaa gtt tat ata gac aga ttt ga#a ttg att cca gtt act     1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605gca aca ttt gaa gca gaa tat gat tta gaa ag#a gca caa aag gcg gtg     1872Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620aat gcg ctg ttt act tct ata aac caa ata gg#g ata aaa aca gat gtg     1920Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl #y Ile Lys Thr Asp Val625                 6 #30                 6 #35                 6 #40acg gat tat cat att gat caa gta tcc aat tt#a gtg gat tgt tta tca     1968Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Asp Cys Leu Ser                645   #               650   #               655gat gaa ttt tgt ctg gat gaa aag cga gaa tt#g tcc gag aaa gtc aaa     2016Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670cat gcg aag cga ctc agt gat gag cgg aat tt#a ctt caa gat cca aac     2064His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Pro Asn        675           #       680           #       685ttc aaa ggc atc aat agg caa cta gac cgt gg#t tgg aga gga agt acg     2112Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl #y Trp Arg Gly Ser Thr    690               #   695               #   700gat att acc atc caa aga gga gat gac gta tt#c aaa gaa aat tat gtc     2160Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20aca cta cca ggt acc ttt gat gag tgc tat cc#a aca tat ttg tat caa     2208Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735aaa atc gat gaa tca aaa tta aaa gcc ttt ac#c cgt tat caa tta aga     2256Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750ggg tat atc gaa gat agt caa gac tta gaa at#c tat tta att cgc tac     2304Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765aat gca aaa cat gaa aca gta aat gtg cca gg#t acg ggt tcc tta tgg     2352Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780ccg ctt tca gcc caa agt cca atc gga aag tg#t gga gag ccg aat cga     2400Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00tgc gcg cca cac ctt gaa tgg aat cct gac tt#a gat tgt tcg tgt agg     2448Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815gat gga gaa aag tgt gcc cat cat tcg cat ca#t ttc tcc tta gac att     2496Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830gat gta gga tgt aca gac tta aat gag gac ct#a ggt gta tgg gtg atc     2544Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845ttt aag att aag acg caa gat ggg cac gca ag#a cta ggg aat cta gag     2592Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860ttt ctc gaa gag aaa cca tta gta gga gaa gc#g cta gct cgt gtg aaa     2640Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80aga gcg gag aaa aaa tgg aga gac aaa cgt ga#a aaa ttg gaa tgg gaa     2688Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895aca aat atc gtt tat aaa gag gca aaa gaa tc#t gta gat gct tta ttt     2736Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910gta aac tct caa tat gat caa tta caa gcg ga#t acg aat att gcc atg     2784Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925att cat gcg gca gat aaa cgt gtt cat agc at#t cga gaa gct tat ctg     2832Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940cct gag ctg tct gtg att ccg ggt gtc aat gc#g gct att ttt gaa gaa     2880Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60tta gaa ggg cgt att ttc act gca ttc tcc ct#a tat gat gcg aga aat     2928Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975gtc att aaa aat ggt gat ttt aat aat ggc tt#a tcc tgc tgg aac gtg     2976Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990aaa ggg cat gta gat gta gaa gaa  caa aac #aac caa cgt  tcg gtc ctt   3024Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005gtt gtt  ccg gaa tgg gaa gca  gaa gtg t#ca caa gaa  gtt cgt gtc      3069Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020tgt ccg  ggt cgt ggc tat atc  ctt cgt g#tc aca gcg  tac aag gag      3114Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035gga tat  gga gaa ggt tgc gta  acc att c#at gag atc  gag aac aat      3159Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050aca gac  gaa ctg aag ttt agc  aac tgc g#ta gaa gag  gaa atc tat      3204Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065cca aat  aac acg gta acg tgt  aat gat t#at act gta  aat caa gaa      3249Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080gaa tac  gga ggt gcg tac act  tct cgt a#at cga gga  tat aac gaa      3294Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095gct cct  tcc gta cca gct gat  tat gcg t#ca gtc tat  gaa gaa aaa      3339Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110tcg tat  aca gat gga cga aga  gag aat c#ct tgt gaa  ttt aac aga      3384Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125ggg tat  agg gat tac acg cca  cta cca g#tt ggt tat  gtg aca aaa      3429Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140gaa tta  gaa tac ttc cca gaa  acc gat a#ag gta tgg  att gag att      3474Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155gga gaa  acg gaa gga aca ttt  atc gtg g#ac agc gtg  gaa tta ctc      3519Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170ctt atg  gag gaa tag           #                   #                  #  3534 Leu Met  Glu Glu     1175 <210> SEQ ID NO 26 <211> LENGTH: 1177<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <400> SEQUENCE: 26Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp His Ala Val        195           #       200           #       205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As #p Ser Arg Thr Tyr Pro                245   #               250   #               255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445Arg Ala Pro Met Phe Ser Trp Thr His Arg Se #r Ala Thr Pro Thr Asn    450               #   455               #   460Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl #y Ile Lys Thr Asp Val625                 6 #30                 6 #35                 6 #40Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Asp Cys Leu Ser                645   #               650   #               655Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Pro Asn        675           #       680           #       685Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl #y Trp Arg Gly Ser Thr    690               #   695               #   700Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170 Leu Met  Glu Glu    1175 <210> SEQ ID NO 27 <211> LENGTH: 3534 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <221> NAME/KEY: CDS<222> LOCATION: (1)..(3531) <400> SEQUENCE: 27atg gat aac aat ccg aac atc aat gaa tgc at#t cct tat aat tgt tta       48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15agt aac cct gaa gta gaa gta tta ggt gga ga#a aga ata gaa act ggt       96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30tac acc cca atc gat att tcc ttg tcg cta ac#g caa ttt ctt ttg agt      144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45gaa ttt gtt ccc ggt gct gga ttt gtg tta gg#a cta gtt gat ata ata      192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60tgg gga att ttt ggt ccc tct caa tgg gac gc#a ttt ctt gta caa att      240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80gaa cag tta att aac caa aga ata gaa gaa tt#c gct agg aac caa gcc      288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95att tct aga tta gaa gga cta agc aat ctt ta#t caa att tac gca gaa      336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110tct ttt aga gag tgg gaa gca gat cct act aa#t cca gca tta aga gaa      384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125gag atg cgt att caa ttc aat gac atg aac ag#t gcc ctt aca acc gct      432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140att cct ctt ttt gca gtt caa aat tat caa gt#t cct ctt tta tca gta      480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60tat gtt caa gct gca aat tta cat tta tca gt#t ttg aga gat gtt tca      528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175gtg ttt gga caa agg tgg gga ttt gat gcc gc#g act atc aat agt cgt      576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190tat aat gat tta act agg ctt att ggc aac ta#t aca gat tat gct gta      624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp Tyr Ala Val        195           #       200           #       205cgc tgg tac aat acg gga tta gaa cgt gta tg#g gga ccg gat tct aga      672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220gat tgg gta agg tat aat caa ttt aga aga ga#a tta aca cta act gta      720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40tta gat atc gtt gct ctg ttc ccg aat tat ga#t agt aga aga tat cca      768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As #p Ser Arg Arg Tyr Pro                245   #               250   #               255att cga aca gtt tcc caa tta aca aga gaa at#t tat aca aac cca gta      816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270tta gaa aat ttt gat ggt agt ttt cga ggc tc#g gct cag ggc ata gaa      864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285aga agt att agg agt cca cat ttg atg gat at#a ctt aac agt ata acc      912Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300atc tat acg gat gct cat agg ggt tat tat ta#t tgg tca ggg cat caa      960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20ata atg gct tct cct gta ggg ttt tcg ggg cc#a gaa ttc act ttt ccg     1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335cta tat gga act atg gga aat gca gct cca ca#a caa cgt att gtt gct     1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350caa cta ggt cag ggc gtg tat aga aca tta tc#g tcc act tta tat aga     1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365aga cct ttt aat ata ggg ata aat aat caa ca#a cta tct gtt ctt gac     1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380ggg aca gaa ttt gct tat gga acc tcc tca aa#t ttg cca tcc gct gta     1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00tac aga aaa agc gga acg gta gat tcg ctg ga#t gaa ata ccg cca cag     1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415aat aac aac gtg cca cct agg caa gga ttt ag#t cat cga tta agc cat     1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430gtt tca atg ttt cgt tca ggc ttt agt aat ag#t agt gta agt ata ata     1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445aga gct cct atg ttc tct tgg ata cat cgt ag#t gct gaa ttt aat aat     1392Arg Ala Pro Met Phe Ser Trp Ile His Arg Se #r Ala Glu Phe Asn Asn    450               #   455               #   460ata att gca tcg gat agt att act caa ata cc#a ttg gta aaa gca cat     1440Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80aca ctt cag tca ggt act act gtt gta aga gg#g ccc ggg ttt acg gga     1488Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495gga gat att ctt cga cga aca agt gga gga cc#a ttt gct tat act att     1536Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510gtt aat ata aat ggg caa tta ccc caa agg ta#t cgt gca aga ata cgc     1584Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525tat gcc tct act aca aat cta aga att tac gt#a acg gtt gca ggt gaa     1632Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540cgg att ttt gct ggt caa ttt aac aaa aca at#g gat acc ggt gac cca     1680Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60tta aca ttc caa tct ttt agt tac gca act at#t aat aca gct ttt aca     1728Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575ttc cca atg agc cag agt agt ttc aca gta gg#t gct gat act ttt agt     1776Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590tca ggg aat gaa gtt tat ata gac aga ttt ga#a ttg att cca gtt act     1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605gca aca ttt gaa gca gaa tat gat tta gaa ag#a gca caa aag gcg gtg     1872Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620aat gcg ctg ttt act tct ata aac caa ata gg#g ata aaa aca gat gtg     1920Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl #y Ile Lys Thr Asp Val625                 6 #30                 6 #35                 6 #40acg gat tat cat att gat caa gta tcc aat tt#a gtg gat tgt tta tca     1968Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Asp Cys Leu Ser                645   #               650   #               655gat gaa ttt tgt ctg gat gaa aag cga gaa tt#g tcc gag aaa gtc aaa     2016Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670cat gcg aag cga ctc agt gat gag cgg aat tt#a ctt caa gat cca aac     2064His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Pro Asn        675           #       680           #       685ttc aaa ggc atc aat agg caa cta gac cgt gg#t tgg aga gga agt acg     2112Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl #y Trp Arg Gly Ser Thr    690               #   695               #   700gat att acc atc caa aga gga gat gac gta tt#c aaa gaa aat tat gtc     2160Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20aca cta cca ggt acc ttt gat gag tgc tat cc#a aca tat ttg tat caa     2208Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735aaa atc gat gaa tca aaa tta aaa gcc ttt ac#c cgt tat caa tta aga     2256Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750ggg tat atc gaa gat agt caa gac tta gaa at#c tat tta att cgc tac     2304Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765aat gca aaa cat gaa aca gta aat gtg cca gg#t acg ggt tcc tta tgg     2352Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780ccg ctt tca gcc caa agt cca atc gga aag tg#t gga gag ccg aat cga     2400Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00tgc gcg cca cac ctt gaa tgg aat cct gac tt#a gat tgt tcg tgt agg     2448Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815gat gga gaa aag tgt gcc cat cat tcg cat ca#t ttc tcc tta gac att     2496Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830gat gta gga tgt aca gac tta aat gag gac ct#a ggt gta tgg gtg atc     2544Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845ttt aag att aag acg caa gat ggg cac gca ag#a cta ggg aat cta gag     2592Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860ttt ctc gaa gag aaa cca tta gta gga gaa gc#g cta gct cgt gtg aaa     2640Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80aga gcg gag aaa aaa tgg aga gac aaa cgt ga#a aaa ttg gaa tgg gaa     2688Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895aca aat atc gtt tat aaa gag gca aaa gaa tc#t gta gat gct tta ttt     2736Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910gta aac tct caa tat gat caa tta caa gcg ga#t acg aat att gcc atg     2784Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925att cat gcg gca gat aaa cgt gtt cat agc at#t cga gaa gct tat ctg     2832Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940cct gag ctg tct gtg att ccg ggt gtc aat gc#g gct att ttt gaa gaa     2880Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60tta gaa ggg cgt att ttc act gca ttc tcc ct#a tat gat gcg aga aat     2928Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975gtc att aaa aat ggt gat ttt aat aat ggc tt#a tcc tgc tgg aac gtg     2976Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990aaa ggg cat gta gat gta gaa gaa  caa aac #aac caa cgt  tcg gtc ctt   3024Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005gtt gtt  ccg gaa tgg gaa gca  gaa gtg t#ca caa gaa  gtt cgt gtc      3069Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020tgt ccg  ggt cgt ggc tat atc  ctt cgt g#tc aca gcg  tac aag gag      3114Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035gga tat  gga gaa ggt tgc gta  acc att c#at gag atc  gag aac aat      3159Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050aca gac  gaa ctg aag ttt agc  aac tgc g#ta gaa gag  gaa atc tat      3204Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065cca aat  aac acg gta acg tgt  aat gat t#at act gta  aat caa gaa      3249Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080gaa tac  gga ggt gcg tac act  tct cgt a#at cga gga  tat aac gaa      3294Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095gct cct  tcc gta cca gct gat  tat gcg t#ca gtc tat  gaa gaa aaa      3339Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110tcg tat  aca gat gga cga aga  gag aat c#ct tgt gaa  ttt aac aga      3384Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125ggg tat  agg gat tac acg cca  cta cca g#tt ggt tat  gtg aca aaa      3429Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140gaa tta  gaa tac ttc cca gaa  acc gat a#ag gta tgg  att gag att      3474Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155gga gaa  acg gaa gga aca ttt  atc gtg g#ac agc gtg  gaa tta ctc      3519Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170ctt atg  gag gaa tag           #                   #                  #  3534 Leu Met  Glu Glu     1175 <210> SEQ ID NO 28 <211> LENGTH: 1177<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <400> SEQUENCE: 28Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp Tyr Ala Val        195           #       200           #       205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As #p Ser Arg Arg Tyr Pro                245   #               250   #               255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445Arg Ala Pro Met Phe Ser Trp Ile His Arg Se #r Ala Glu Phe Asn Asn    450               #   455               #   460Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gl #y Ile Lys Thr Asp Val625                 6 #30                 6 #35                 6 #40Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Asp Cys Leu Ser                645   #               650   #               655Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Pro Asn        675           #       680           #       685Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gl #y Trp Arg Gly Ser Thr    690               #   695               #   700Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170 Leu Met  Glu Glu    1175 <210> SEQ ID NO 29 <211> LENGTH: 3579 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <400> SEQUENCE: 29atggataaca atccgaacat caatgaatgc attccttata attgtttaag ta#accctgaa     60gtagaagtat taggtggaga aagaatagaa actggttaca ccccaatcga ta#tttccttg    120tcgctaacgc aatttctttt gagtgaattt gttcccggtg ctggatttgt gt#taggacta    180gttgatataa tatggggaat ttttggtccc tctcaatggg acgcatttct tg#tacaaatt    240gaacagttaa ttaaccaaag aatagaagaa ttcgctagga accaagccat tt#ctagatta    300gaaggactaa gcaatcttta tcaaatttac gcagaatctt ttagagagtg gg#aagcagat    360cctactaatc cagcattaag agaagagatg cgtattcaat tcaatgacat ga#acagtgcc    420cttacaaccg ctattcctct ttttgcagtt caaaattatc aagttcctct tt#tatcagta    480tatgttcaag ctgcaaattt acatttatca gttttgagag atgtttcagt gt#ttggacaa    540aggtggggat ttgatgccgc gactatcaat agtcgttata atgatttaac ta#ggcttatt    600ggcaactata cagattatgc tgtacgctgg tacaatacgg gattagaacg tg#tatgggga    660ccggattcta gagattgggt aaggtataat caatttagaa gagaattaac ac#taactgta    720ttagatatcg ttgctctgtt cccgaattat gatagtagaa gatatccaat tc#gaacagtt    780tcccaattaa caagagaaat ttatacaaac ccagtattag aaaattttga tg#gtagtttt    840cgaggctcgg ctcagggcat agaaagaagt attaggagtc cacatttgat gg#atatactt    900aacagtataa ccatctatac ggatgctcat aggggttatt attattggtc ag#ggcatcaa    960ataatggctt ctcctgtagg gttttcgggg ccagaattca cttttccgct at#atggaact   1020atgggaaatg cagctccaca acaacgtatt gttgctcaac taggtcaggg cg#tgtataga   1080acattatcgt ccactttata tagaagacct tttaatatag ggataaataa tc#aacaacta   1140tctgttcttg acgggacaga atttgcttat ggaacctcct caaatttgcc at#ccgctgta   1200tacagaaaaa gcggaacggt agattcgctg gatgaaatac cgccacagaa ta#acaacgtg   1260ccacctaggc aaggatttag tcatcgatta agccatgttt caatgtttcg tt#caggcttt   1320agtaatagta gtgtaagtat aataagagct cctatgttct cttggataca tc#gtagtgca   1380actcttacaa atacaattga tccagagaga attaatcaaa tacctttagt ga#aaggattt   1440agagtttggg ggggcacctc tgtcattaca ggaccaggat ttacaggagg gg#atatcctt   1500cgaagaaata cctttggtga ttttgtatct ctacaagtca atattaattc ac#caattacc   1560caaagatacc gtttaagatt tcgttacgct tccagtaggg atgcacgagt ta#tagtatta   1620acaggagcgg catccacagg agtgggaggc caagttagtg taaatatgcc tc#ttcagaaa   1680actatggaaa taggggagaa cttaacatct agaacattta gatataccga tt#ttagtaat   1740cctttttcat ttagagctaa tccagatata attgggataa gtgaacaacc tc#tatttggt   1800gcaggttcta ttagtagcgg tgaactttat atagataaaa ttgaaattat tc#tagcagat   1860gcaacatttg aagcagaatc tgatttagaa agagcacaaa aggcggtgaa tg#ccctgttt   1920acttcttcca atcaaatcgg gttaaaaacc gatgtgacgg attatcatat tg#atcaagta   1980tccaatttag tggattgttt atcagatgaa ttttgtctgg atgaaaagcg ag#aattgtcc   2040gagaaagtca aacatgcgaa gcgactcagt gatgagcgga atttacttca ag#atccaaac   2100ttcagaggga tcaatagaca accagaccgt ggctggagag gaagtacaga ta#ttaccatc   2160caaggaggag atgacgtatt caaagagaat tacgtcacac taccgggtac cg#ttgatgag   2220tgctatccaa cgtatttata tcagaaaata gatgagtcga aattaaaagc tt#atacccgt   2280tatgaattaa gagggtatat cgaagatagt caagacttag aaatctattt ga#tccgttac   2340aatgcaaaac acgaaatagt aaatgtgcca ggcacgggtt ccttatggcc gc#tttcagcc   2400caaagtccaa tcggaaagtg tggagaaccg aatcgatgcg cgccacacct tg#aatggaat   2460cctgatctag attgttcctg cagagacggg gaaaaatgtg cacatcattc cc#atcatttc   2520accttggata ttgatgttgg atgtacagac ttaaatgagg acttaggtgt at#gggtgata   2580ttcaagatta agacgcaaga tggccatgca agactaggga atctagagtt tc#tcgaagag   2640aaaccattat taggggaagc actagctcgt gtgaaaagag cggagaagaa gt#ggagagac   2700aaacgagaga aactgcagtt ggaaacaaat attgtttata aagaggcaaa ag#aatctgta   2760gatgctttat ttgtaaactc tcaatatgat agattacaag tggatacgaa ca#tcgcaatg   2820attcatgcgg cagataaacg cgttcataga atccgggaag cgtatctgcc ag#agttgtct   2880gtgattccag gtgtcaatgc ggccattttc gaagaattag agggacgtat tt#ttacagcg   2940tattccttat atgatgcgag aaatgtcatt aaaaatggcg atttcaataa tg#gcttatta   3000tgctggaacg tgaaaggtca tgtagatgta gaagagcaaa acaaccaccg tt#cggtcctt   3060gttatcccag aatgggaggc agaagtgtca caagaggttc gtgtctgtcc ag#gtcgtggc   3120tatatccttc gtgtcacagc atataaagag ggatatggag agggctgcgt aa#cgatccat   3180gagatcgaag acaatacaga cgaactgaaa ttcagcaact gtgtagaaga gg#aagtatat   3240ccaaacaaca cagtaacgtg taataattat actgggactc aagaagaata tg#agggtacg   3300tacacttctc gtaatcaagg atatgacgaa gcctatggta ataacccttc cg#taccagct   3360gattacgctt cagtctatga agaaaaatcg tatacagatg gacgaagaga ga#atccttgt   3420gaatctaaca gaggctatgg ggattacaca ccactaccgg ctggttatgt aa#caaaggat   3480ttagagtact tcccagagac cgataaggta tggattgaga tcggagaaac ag#aaggaaca   3540 ttcatcgtgg atagcgtgga attactcctt atggaggaa      #                   #  3579 <210> SEQ ID NO 30 <211> LENGTH: 1193<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <400> SEQUENCE: 30Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp Tyr Ala Val        195           #       200           #       205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As #p Ser Arg Arg Tyr Pro                245   #               250   #               255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445Arg Ala Pro Met Phe Ser Trp Ile His Arg Se #r Ala Thr Leu Thr Asn    450               #   455               #   460Thr Ile Asp Pro Glu Arg Ile Asn Gln Ile Pr #o Leu Val Lys Gly Phe465                 4 #70                 4 #75                 4 #80Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly As #p Phe Val Ser Leu Gln            500       #           505       #           510Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Ty #r Arg Leu Arg Phe Arg        515           #       520           #       525Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Va #l Leu Thr Gly Ala Ala    530               #   535               #   540Ser Thr Gly Val Gly Gly Gln Val Ser Val As #n Met Pro Leu Gln Lys545                 5 #50                 5 #55                 5 #60Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Ar #g Thr Phe Arg Tyr Thr                565   #               570   #               575Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala As #n Pro Asp Ile Ile Gly            580       #           585       #           590Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Se #r Ile Ser Ser Gly Glu        595           #       600           #       605Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Al #a Asp Ala Thr Phe Glu    610               #   615               #   620Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Al #a Val Asn Ala Leu Phe625                 6 #30                 6 #35                 6 #40Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr As #p Val Thr Asp Tyr His                645   #               650   #               655Ile Asp Gln Val Ser Asn Leu Val Asp Cys Le #u Ser Asp Glu Phe Cys            660       #           665       #           670Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Va #l Lys His Ala Lys Arg        675           #       680           #       685Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pr #o Asn Phe Arg Gly Ile    690               #   695               #   700Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Se #r Thr Asp Ile Thr Ile705                 7 #10                 7 #15                 7 #20Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Ty #r Val Thr Leu Pro Gly                725   #               730   #               735Thr Val Asp Glu Cys Tyr Pro Thr Tyr Leu Ty #r Gln Lys Ile Asp Glu            740       #           745       #           750Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Le #u Arg Gly Tyr Ile Glu        755           #       760           #       765Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Ar #g Tyr Asn Ala Lys His    770               #   775               #   780Glu Ile Val Asn Val Pro Gly Thr Gly Ser Le #u Trp Pro Leu Ser Ala785                 7 #90                 7 #95                 8 #00Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro As #n Arg Cys Ala Pro His                805   #               810   #               815Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cy #s Arg Asp Gly Glu Lys            820       #           825       #           830Cys Ala His His Ser His His Phe Thr Leu As #p Ile Asp Val Gly Cys        835           #       840           #       845Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Va #l Ile Phe Lys Ile Lys    850               #   855               #   860Thr Gln Asp Gly His Ala Arg Leu Gly Asn Le #u Glu Phe Leu Glu Glu865                 8 #70                 8 #75                 8 #80Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Va #l Lys Arg Ala Glu Lys                885   #               890   #               895Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Le #u Glu Thr Asn Ile Val            900       #           905       #           910Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Le #u Phe Val Asn Ser Gln        915           #       920           #       925Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Al #a Met Ile His Ala Ala    930               #   935               #   940Asp Lys Arg Val His Arg Ile Arg Glu Ala Ty #r Leu Pro Glu Leu Ser945                 9 #50                 9 #55                 9 #60Val Ile Pro Gly Val Asn Ala Ala Ile Phe Gl #u Glu Leu Glu Gly Arg                965   #               970   #               975Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Ar #g Asn Val Ile Lys Asn            980       #           985       #           990Gly Asp Phe Asn Asn Gly Leu Leu  Cys Trp  #Asn Val Lys  Gly His Val        995           #       1000           #       1005Asp Val  Glu Glu Gln Asn Asn  His Arg S #er Val Leu  Val Ile Pro    1010              #    1015              #    1020Glu Trp  Glu Ala Glu Val Ser  Gln Glu V #al Arg Val  Cys Pro Gly    1025              #    1030              #    1035Arg Gly  Tyr Ile Leu Arg Val  Thr Ala T #yr Lys Glu  Gly Tyr Gly    1040              #    1045              #    1050Glu Gly  Cys Val Thr Ile His  Glu Ile G #lu Asp Asn  Thr Asp Glu    1055              #    1060              #    1065Leu Lys  Phe Ser Asn Cys Val  Glu Glu G #lu Val Tyr  Pro Asn Asn    1070              #    1075              #    1080Thr Val  Thr Cys Asn Asn Tyr  Thr Gly T #hr Gln Glu  Glu Tyr Glu    1085              #    1090              #    1095Gly Thr  Tyr Thr Ser Arg Asn  Gln Gly T #yr Asp Glu  Ala Tyr Gly    1100              #    1105              #    1110Asn Asn  Pro Ser Val Pro Ala  Asp Tyr A #la Ser Val  Tyr Glu Glu    1115              #    1120              #    1125Lys Ser  Tyr Thr Asp Gly Arg  Arg Glu A #sn Pro Cys  Glu Ser Asn    1130              #    1135              #    1140Arg Gly  Tyr Gly Asp Tyr Thr  Pro Leu P #ro Ala Gly  Tyr Val Thr    1145              #    1150              #    1155Lys Asp  Leu Glu Tyr Phe Pro  Glu Thr A #sp Lys Val  Trp Ile Glu    1160              #    1165              #    1170Ile Gly  Glu Thr Glu Gly Thr  Phe Ile V #al Asp Ser  Val Glu Leu    1175              #    1180              #    1185Leu Leu  Met Glu Glu     1190 <210> SEQ ID NO 31 <211> LENGTH: 16<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 31cgttgctctg ttcccg              #                   #                  #    16 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 32tcaaatacca ttggtaaaag             #                  #                   # 20 <210> SEQ ID NO 33 <211> LENGTH: 3534<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <400> SEQUENCE: 33atggataaca atccgaacat caatgaatgc attccttata attgtttaag ta#accctgaa     60gtagaagtat taggtggaga aagaatagaa actggttaca ccccaatcga ta#tttccttg    120tcgctaacgc aatttctttt gagtgaattt gttcccggtg ctggatttgt gt#taggacta    180gttgatataa tatggggaat ttttggtccc tctcaatggg acgcatttct tg#tacaaatt    240gaacagttaa ttaaccaaag aatagaagaa ttcgctagga accaagccat tt#ctagatta    300gaaggactaa gcaatcttta tcaaatttac gcagaatctt ttagagagtg gg#aagcagat    360cctactaatc cagcattaag agaagagatg cgtattcaat tcaatgacat ga#acagtgcc    420cttacaaccg ctattcctct ttttgcagtt caaaattatc aagttcctct tt#tatcagta    480tatgttcaag ctgcaaattt acatttatca gttttgagag atgtttcagt gt#ttggacaa    540aggtggggat ttgatgccgc gactatcaat agtcgttata atgatttaac ta#ggcttatt    600ggcaactata cagattatgc tgtacgctgg tacaatacgg gattagaacg tg#tatgggga    660ccggattcta gagattgggt aaggtataat caatttagaa gagaattaac ac#taactgta    720ttagatatcg ttgctctgtt cccgaattat gatagtagaa gatatccaat tc#gaacagtt    780tcccaattaa caagagaaat ttatacaaac ccagtattag aaaattttga tg#gtagtttt    840cgaggctcgg ctcagggcat agaaagaagt attaggagtc cacatttgat gg#atatactt    900aacagtataa ccatctatac ggatgctcat aggggttatt attattggtc ag#ggcatcaa    960ataatggctt ctcctgtagg gttttcgggg ccagaattca cttttccgct at#atggaact   1020atgggaaatg cagctccaca acaacgtatt gttgctcaac taggtcaggg cg#tgtataga   1080acattatcgt ccactttata tagaagacct tttaatatag ggataaataa tc#aacaacta   1140tctgttcttg acgggacaga atttgcttat ggaacctcct caaatttgcc at#ccgctgta   1200tacagaaaaa gcggaacggt agattcgctg gatgaaatac cgccacagaa ta#acaacgtg   1260ccacctaggc aaggatttag tcatcgatta agccatgttt caatgtttcg tt#caggcttt   1320agtaatagta gtgtaagtat aataagagct cctatgttct cttggataca tc#gtagtgct   1380gaatttaata atataattgc atcggatagt attactcaaa taccattggt aa#aagcacat   1440acacttcagt caggtactac tgttgtaaga gggcccgggt ttacgggagg ag#atattctt   1500cgacgaacaa gtggaggacc atttgcttat actattgtta atataaatgg gc#aattaccc   1560caaaggtatc gtgcaagaat acgctatgcc tctactacaa atctaagaat tt#acgtaacg   1620gttgcaggtg aacggatttt tgctggtcaa tttaacaaaa caatggatac cg#gtgaccca   1680ttaacattcc aatcttttag ttacgcaact attaatacag cttttacatt cc#caatgagc   1740cagagtagtt tcacagtagg tgctgatact tttagttcag ggaatgaagt tt#atatagac   1800agatttgaat tgattccagt tactgcaaca ctcgaggctg aatataatct gg#aaagagcg   1860cagaaggcgg tgaatgcgct gtttacgtct acaaaccaac tagggctaaa aa#caaatgta   1920acggattatc atattgatca agtgtccaat ttagttacgt atttatcgga tg#aattttgt   1980ctggatgaaa agcgagaatt gtccgagaaa gtcaaacatg cgaagcgact ca#gtgatgaa   2040cgcaatttac tccaagattc aaatttcaaa gacattaata ggcaaccaga ac#gtgggtgg   2100ggcggaagta cagggattac catccaagga ggggatgacg tatttaaaga aa#attacgtc   2160acactatcag gtacctttga tgagtgctat ccaacatatt tgtatcaaaa aa#tcgatgaa   2220tcaaaattaa aagcctttac ccgttatcaa ttaagagggt atatcgaaga ta#gtcaagac   2280ttagaaatct atttaattcg ctacaatgca aaacatgaaa cagtaaatgt gc#caggtacg   2340ggttccttat ggccgctttc agcccaaagt ccaatcggaa agtgtggaga gc#cgaatcga   2400tgcgcgccac accttgaatg gaatcctgac ttagattgtt cgtgtaggga tg#gagaaaag   2460tgtgcccatc attcgcatca tttctcctta gacattgatg taggatgtac ag#acttaaat   2520gaggacctag gtgtatgggt gatctttaag attaagacgc aagatgggca cg#caagacta   2580gggaatctag agtttctcga agagaaacca ttagtaggag aagcgctagc tc#gtgtgaaa   2640agagcggaga aaaaatggag agacaaacgt gaaaaattgg aatgggaaac aa#atatcgtt   2700tataaagagg caaaagaatc tgtagatgct ttatttgtaa actctcaata tg#atcaatta   2760caagcggata cgaatattgc catgattcat gcggcagata aacgtgttca ta#gcattcga   2820gaagcttatc tgcctgagct gtctgtgatt ccgggtgtca atgcggctat tt#ttgaagaa   2880ttagaagggc gtattttcac tgcattctcc ctatatgatg cgagaaatgt ca#ttaaaaat   2940ggtgatttta ataatggctt atcctgctgg aacgtgaaag ggcatgtaga tg#tagaagaa   3000caaaacaacc aacgttcggt ccttgttgtt ccggaatggg aagcagaagt gt#cacaagaa   3060gttcgtgtct gtccgggtcg tggctatatc cttcgtgtca cagcgtacaa gg#agggatat   3120ggagaaggtt gcgtaaccat tcatgagatc gagaacaata cagacgaact ga#agtttagc   3180aactgcgtag aagaggaaat ctatccaaat aacacggtaa cgtgtaatga tt#atactgta   3240aatcaagaag aatacggagg tgcgtacact tctcgtaatc gaggatataa cg#aagctcct   3300tccgtaccag ctgattatgc gtcagtctat gaagaaaaat cgtatacaga tg#gacgaaga   3360gagaatcctt gtgaatttaa cagagggtat agggattaca cgccactacc ag#ttggttat   3420gtgacaaaag aattagaata cttcccagaa accgataagg tatggattga ga#ttggagaa   3480acggaaggaa catttatcgt ggacagcgtg gaattactcc ttatggagga at#ag         3534 <210> SEQ ID NO 34 <211> LENGTH: 1177 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Hybrid Delta-Endotoxin <400> SEQUENCE: 34Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il #e Pro Tyr Asn Cys Leu1               5    #                10   #                15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl #u Arg Ile Glu Thr Gly            20       #            25       #            30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th #r Gln Phe Leu Leu Ser        35           #        40           #        45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl #y Leu Val Asp Ile Ile    50               #    55               #    60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al #a Phe Leu Val Gln Ile65                   #70                   #75                   #80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph #e Ala Arg Asn Gln Ala                85   #                90   #                95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty #r Gln Ile Tyr Ala Glu            100       #           105       #           110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As #n Pro Ala Leu Arg Glu        115           #       120           #       125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se #r Ala Leu Thr Thr Ala    130               #   135               #   140Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va #l Pro Leu Leu Ser Val145                 1 #50                 1 #55                 1 #60Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va #l Leu Arg Asp Val Ser                165   #               170   #               175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al #a Thr Ile Asn Ser Arg            180       #           185       #           190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty #r Thr Asp Tyr Ala Val        195           #       200           #       205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr #p Gly Pro Asp Ser Arg    210               #   215               #   220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl #u Leu Thr Leu Thr Val225                 2 #30                 2 #35                 2 #40Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As #p Ser Arg Arg Tyr Pro                245   #               250   #               255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il #e Tyr Thr Asn Pro Val            260       #           265       #           270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se #r Ala Gln Gly Ile Glu        275           #       280           #       285Arg Ser Ile Arg Ser Pro His Leu Met Asp Il #e Leu Asn Ser Ile Thr    290               #   295               #   300Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty #r Trp Ser Gly His Gln305                 3 #10                 3 #15                 3 #20Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr #o Glu Phe Thr Phe Pro                325   #               330   #               335Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl #n Gln Arg Ile Val Ala            340       #           345       #           350Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se #r Ser Thr Leu Tyr Arg        355           #       360           #       365Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl #n Leu Ser Val Leu Asp    370               #   375               #   380Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As #n Leu Pro Ser Ala Val385                 3 #90                 3 #95                 4 #00Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As #p Glu Ile Pro Pro Gln                405   #               410   #               415Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se #r His Arg Leu Ser His            420       #           425       #           430Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se #r Ser Val Ser Ile Ile        435           #       440           #       445Arg Ala Pro Met Phe Ser Trp Ile His Arg Se #r Ala Glu Phe Asn Asn    450               #   455               #   460Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pr #o Leu Val Lys Ala His465                 4 #70                 4 #75                 4 #80Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gl #y Pro Gly Phe Thr Gly                485   #               490   #               495Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pr #o Phe Ala Tyr Thr Ile            500       #           505       #           510Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Ty #r Arg Ala Arg Ile Arg        515           #       520           #       525Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Va #l Thr Val Ala Gly Glu    530               #   535               #   540Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Me #t Asp Thr Gly Asp Pro545                 5 #50                 5 #55                 5 #60Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Il #e Asn Thr Ala Phe Thr                565   #               570   #               575Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gl #y Ala Asp Thr Phe Ser            580       #           585       #           590Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Gl #u Leu Ile Pro Val Thr        595           #       600           #       605Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Ar #g Ala Gln Lys Ala Val    610               #   615               #   620Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gl #y Leu Lys Thr Asn Val625                 6 #30                 6 #35                 6 #40Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le #u Val Thr Tyr Leu Ser                645   #               650   #               655Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Le #u Ser Glu Lys Val Lys            660       #           665       #           670His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le #u Leu Gln Asp Ser Asn        675           #       680           #       685Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gl #y Trp Gly Gly Ser Thr    690               #   695               #   700Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Ph #e Lys Glu Asn Tyr Val705                 7 #10                 7 #15                 7 #20Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pr #o Thr Tyr Leu Tyr Gln                725   #               730   #               735Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Th #r Arg Tyr Gln Leu Arg            740       #           745       #           750Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il #e Tyr Leu Ile Arg Tyr        755           #       760           #       765Asn Ala Lys His Glu Thr Val Asn Val Pro Gl #y Thr Gly Ser Leu Trp    770               #   775               #   780Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cy #s Gly Glu Pro Asn Arg785                 7 #90                 7 #95                 8 #00Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le #u Asp Cys Ser Cys Arg                805   #               810   #               815Asp Gly Glu Lys Cys Ala His His Ser His Hi #s Phe Ser Leu Asp Ile            820       #           825       #           830Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le #u Gly Val Trp Val Ile        835           #       840           #       845Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar #g Leu Gly Asn Leu Glu    850               #   855               #   860Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al #a Leu Ala Arg Val Lys865                 8 #70                 8 #75                 8 #80Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl #u Lys Leu Glu Trp Glu                885   #               890   #               895Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se #r Val Asp Ala Leu Phe            900       #           905       #           910Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala As #p Thr Asn Ile Ala Met        915           #       920           #       925Ile His Ala Ala Asp Lys Arg Val His Ser Il #e Arg Glu Ala Tyr Leu    930               #   935               #   940Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al #a Ala Ile Phe Glu Glu945                 9 #50                 9 #55                 9 #60Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le #u Tyr Asp Ala Arg Asn                965   #               970   #               975Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le #u Ser Cys Trp Asn Val            980       #           985       #           990Lys Gly His Val Asp Val Glu Glu  Gln Asn  #Asn Gln Arg  Ser Val Leu        995           #       1000           #       1005Val Val  Pro Glu Trp Glu Ala  Glu Val S #er Gln Glu  Val Arg Val    1010              #    1015              #    1020Cys Pro  Gly Arg Gly Tyr Ile  Leu Arg V #al Thr Ala  Tyr Lys Glu    1025              #    1030              #    1035Gly Tyr  Gly Glu Gly Cys Val  Thr Ile H #is Glu Ile  Glu Asn Asn    1040              #    1045              #    1050Thr Asp  Glu Leu Lys Phe Ser  Asn Cys V #al Glu Glu  Glu Ile Tyr    1055              #    1060              #    1065Pro Asn  Asn Thr Val Thr Cys  Asn Asp T #yr Thr Val  Asn Gln Glu    1070              #    1075              #    1080Glu Tyr  Gly Gly Ala Tyr Thr  Ser Arg A #sn Arg Gly  Tyr Asn Glu    1085              #    1090              #    1095Ala Pro  Ser Val Pro Ala Asp  Tyr Ala S #er Val Tyr  Glu Glu Lys    1100              #    1105              #    1110Ser Tyr  Thr Asp Gly Arg Arg  Glu Asn P #ro Cys Glu  Phe Asn Arg    1115              #    1120              #    1125Gly Tyr  Arg Asp Tyr Thr Pro  Leu Pro V #al Gly Tyr  Val Thr Lys    1130              #    1135              #    1140Glu Leu  Glu Tyr Phe Pro Glu  Thr Asp L #ys Val Trp  Ile Glu Ile    1145              #    1150              #    1155Gly Glu  Thr Glu Gly Thr Phe  Ile Val A #sp Ser Val  Glu Leu Leu    1160              #    1165              #    1170 Leu Met  Glu Glu    1175 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 35tgcaacactc gaggctgaat             #                  #                   # 20

What is claimed is:
 1. A purified antibody generated by using apolypeptide comprising SEQ ID NO: 10 as an immunogen.
 2. Animmunodetection kit comprising, in suitable container means, theantibody according to claim 1, and an immunodetection reagent.
 3. Thepurified antibody of claim 1, operably attached to a detectable label.